Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area,Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, and salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54°C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected in the samples from both the thermophilic and mesophilic mats. Cultures of nonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolated from the mats developed at high (50.6–49.4°C) and low temperatures (45–20°C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealed in low-temperature mats. Truly thermophilic purple and green sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfur communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophyll a-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20°C) mat is of interest.     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

Alkaliphilic sulfidogenesis on cellulose by combined cultures

Soda lakes are characterized by an intense sulfur cycle that begins with sulfidogenesis. Model laboratory experiments that involved combining of pure cultures showed that, during anaerobic decomposition of cellulose by Clostridium alkalicellulosi, the sulfate-reducing bacteria (SRB) of the species Desulfonatronovibrio hydrogenovorans, Desulfonatronum lacustre, and Desulfonatronum cooperativum, different in their nutritional requirements, may directly use the cellulose fermentation products for sulfidogenesis without mediatory microorganisms. In binary cocultures with SRB, the amount of the H₂S formed constituted from one-third to two-thirds of the cellulose [H] equivalents; acetate was among the products formed. When the syntrophic Contubernalis alkalaceticum, capable of acetate oxidation, was incorporated into the trophic chain along with hydrogenotrophic SRB, the amount of the H₂S formed exceeded by 33–42% the amount of the [H] equivalents in the utilized cellulose, water being the source of additional hydrogen. Thus, the trophic pathway from plant residues to sulfide, previously considered to be the longest in the alkaliphilic microbial community, may involve a minimal number of stages and do without intermediate participation of dissipotrophic fermenting organisms.     

MAPKs as a cross point in H2O2 and auxin signaling under combined cadmium and zinc stress in rice roots

Previously, we have reported the role of MAPKs (mitogen-activated protein kinases) under cadmium stress. This work continue to explore the relationship between MAPKs, H₂O₂, auxin signaling, and OsHMA and OsZIP gene expression in rice (Oryza sativa L.) roots under combined cadmium (Cd) and zinc (Zn) stress. Compared with Cd, Cd+Zn reduced Cd levels but increased Zn accumulation in the roots. Three OsMAPK genes were negatively regulated, while two OsHMA and two OsZIP genes were positively regulated by MAPK pathways under Cd+Zn stress. Transgenic rice expressing DR5-GUS exhibited enhanced GUS activity in H₂O₂-, PD (MAPKK inhibitor PD98059)-, or (Cd+Zn)-treated roots, which also exhibited increased H₂O₂ concentrations, whereas GUS staining decreased in roots in response to Cd+Zn+PD, DMTU (N,N′-dimethylthiourea, a H₂O₂ scavenger), or Cd+Zn+DMTU treatment, with reduced H₂O₂ levels. GUS levels were consistent with H₂O₂ levels, suggesting that MAPK pathway-mediated auxin redistribution occurs via H₂O₂, and H₂O₂ functions downstream of MAPK but upstream of auxin signaling pathways. Furthermore, MAPK pathways serve specific functions in regulating the expression of some key genes of auxin signaling (OsYUCCA, OsPIN, OsARF, and OsIAA) under Cd+Zn stress. Overall, MAPK cascades function in the integration of metal transport, H₂O₂ generation, and auxin signaling in rice seedlings grown under Cd+Zn stress.     

Rhodopseudomonas globiformis,sp. n., a new species of the Rhodospirillaceae

Enrichments of pH: 5.1, inoculated from a warm, acidic sulfur spring of the Yellowstone Park, yielded purple red cultures of a mobile, spherical organism belonging to the Rhodospirillaceae, described herein as a new species Rhodopseudomonas globiformis. Under favourable conditions, the cells are 1.6–1.8 μm in diameter, smaller and larger cells may occur. The photopigments consist of bacterio-chlorophyll a_P and new aliphatic methoxylated ketocarotenoids. The organism grows either under anaerobic conditions in the light or under microaerophilic conditions in the dark. Biotin, p-aminobenzoic acid and a source of reduced sulfur are required as growth factors. Gluconate, mannitol, fructose and ethanol are the best carbon sources at a pH of 4.9. Growth is inhibited by low concentrations of sulfide.     

Control of Oxidative Sulfur Metabolism of Chlorobium limicola forma thiosulfatophilum

A metered blend of anaerobic-grade N₂, CO₂, and H₂S gases was introduced into an illuminated, 800-ml liquid volume, continuously stirred tank reactor. The system, described as an anaerobic gas-to-liquid phase fed-batch reactor, was used to investigate the effects of H₂S flow rate and light energy on the accumulation of oxidized sulfur compounds formed by the photoautotroph Chlorobium limicola forma thiosulfatophilum during growth. Elemental sulfur was formed and accumulated in stoichiometric quantities when light energy and H₂S molar flow rate levels were optimally adjusted in the presence of nonlimiting CO₂. Deviation from the optimal H₂S and light energy levels resulted in either oxidation of sulfur or complete inhibition of sulfide oxidation. Based on these observations, a model of sulfide and sulfur oxidases electrochemically coupled to the photosynthetic reaction center of Chlorobium spp. is presented. The dynamic deregulation of oxidative pathways may be a mechanism for supplying the photosynthetic reaction center with a continuous source of electrons during periods of varying light and substrate availability, as in pond ecosystems where Chlorobium spp. are found. Possible applications for a sulfide gas removal process are discussed.     

H<Subscript>2</Subscript>S biotreatment with sulfide-oxidizing heterotrophic bacteria

Many industrial activities produce H₂S, which is toxic at high levels and odorous at even very low levels. Chemolithotrophic sulfur-oxidizing bacteria are often used in its remediation. Recently, we have reported that many heterotrophic bacteria can use sulfide:quinone oxidoreductase and persulfide dioxygenase to oxidize H₂S to thiosulfate and sulfite. These bacteria may also potentially be used in H₂S biotreatment. Here we report how various heterotrophic bacteria with these enzymes were cultured with organic compounds and the cells were able to rapidly oxidize H₂S to zero-valence sulfur and thiosulfate, causing no apparent acidification. Some also converted the produced thiosulfate to tetrathionate. The rates of sulfide oxidation by some of the tested bacteria in suspension, ranging from 8 to 50 µmol min^?1 g^?1 of cell dry weight at pH 7.4, sufficient for H₂S biotreatment. The immobilized bacteria removed H₂S as efficiently as the bacteria in suspension, and the inclusion of Fe₃O₄ nanoparticles during immobilization resulted in increased efficiency for sulfide removal, in part due to chemical oxidation H₂S by Fe₃O₄. Thus, heterotrophic bacteria may be used for H₂S biotreatment under aerobic conditions.     

Lipopolysaccharide enhances the cytotoxicity of 2-chloroethyl ethyl sulfide

Background

The bacterial endotoxin, lipopolysaccharide (LPS), is a well-characterized inflammatory factor found in the cell wall of Gram-negative bacteria. In this investigation, we studied the cytotoxic interaction between 2-chloroethyl ethyl sulfide (CEES or ClCH₂CH₂SCH₂CH₃) and LPS using murine RAW264.7 macrophages. CEES is a sulfur vesicating agent and is an analog of 2,2'-dichlorodiethyl sulfide (sulfur mustard). LPS is a ubiquitous natural agent found in the environment. The ability of LPS and other inflammatory agents (such as TNF-alpha and IL-1beta) to modulate the toxicity of CEES is likely to be an important factor in the design of effective treatments.

Results

RAW 264.7 macrophages stimulated with LPS were found to be more susceptible to the cytotoxic effect of CEES than unstimulated macrophages. Very low levels of LPS (20 ng/ml) dramatically enhanced the toxicity of CEES at concentrations greater than 400 μM. The cytotoxic interaction between LPS and CEES reached a maximum 12 hours after exposure. In addition, we found that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) as well as phorbol myristate acetate (PMA) also enhanced the cytotoxic effects of CEES but to a lesser extent than LPS.

Conclusion

Our in vitro results suggest the possibility that LPS and inflammatory cytokines could enhance the toxicity of sulfur mustard. Since LPS is a ubiquitous agent in the natural environment, its presence is likely to be an important variable influencing the cytotoxicity of sulfur mustard toxicity. We have initiated further experiments to determine the molecular mechanism whereby the inflammatory process influences sulfur mustard cytotoxicity.

     

Distribution of iron in Lake Banyoles in relation to the ecology of purple and green sulfur bacteria

The distribution of iron both in suspended sediment and in the water column has been studied during summer stratification in Lake Banyoles. In this lake, near bottom springs, a very fine material suspended sediment remains in suspension. Dissolved Fe²⁺ in interstitial water of this suspended sediment, is related to redox potential and to the bottom water inflow. In the water column, soluble iron is present in the hypolimnion of the six different basins forming Lake Banyoles. Under those conditions Fe²⁺ is partially removed by sulfide produced in the anoxic sediment. In addition, a peak of Fe²⁺ found at the density gradient level in basins C-III, C-IV and C-VI. A three compartment model on the dynamics of the processes involving iron in Lake Banyoles is proposed. The bottom springs supply oxygen to the anoxic hypolimnion affecting chemical processes of the iron cycle. The presence of phototrophic sulfur bacteria in the anoxic monimolimnion of basins C-III and C-IV can be related to the kinetics of Fe²⁺ and sulfide. In C-III sulfide concentration exceeds Fe²⁺ concentration whereas in C-IV sulfide is not detectable and iron reached values up to 60 mM. The presence of phototrophic sulfur bacteria in iron-containing environments with no detectable sulfide is explained by the ability of such microorganisms to use FeS as electron donor instead of H₂S.     

Effects of sulfur-metabolizing bacterial community diversity on H<Subscript>2</Subscript>S emission behavior in landfills with different operation modes

Hydrogen sulfide (H₂S) is one of the major contributors to offensive odors from landfills, and its concentration differs under different operation modes. This study examined the distribution of H₂S emission from different landfill depths under different operation modes (anaerobic, semi-aerobic, semi-aerobic transformation, and the three operation modes with additional leachate recirculation). The microbial community (especially the sulfur-metabolizing bacterial community) was investigated using high-throughput sequencing technology. The results showed that the semi-aerobic mode could substantially lower the risks of H₂S pollution in landfills, which might be because of the difference in biological processes related to sulfur metabolism driven by functional microbes. A myriad of factors are responsible for mutually shaping the sulfur-metabolizing bacterial community composition in landfills that might subsequently affect the behavior of H₂S emission in landfills. The differences in abundance of the genera Acinetobacter and Paracoccus (phylum Proteobacteria) caused by environmental factors might explain the differences in H₂S emission. H₂S odor control could be realized if the related functional microbe diversity can be influenced by adjustments to landfill operation.     

Pseudomonas aeruginosa MdaB and WrbA are water-soluble two-electron quinone oxidoreductases with the potential to defend against oxidative stress

Water-soluble quinone oxidoreductases capable of reducing quinone substrates via a concerted two-electron mechanism have been implicated in bacterial antioxidant defence. Twoelectron transfer avoids formation of dangerously reactive semi-quinone intermediates, moreover previous work in Pseudomonas putida indicated a direct protective effect for the quinols generated by an over-expressed oxidoreductase. Here, the Pseudomonas aeruginosa orthologs of five quinone oxidoreductases — MdaB, ChrR, WrbA, NfsB, and NQO1 — were tested for their possible role in defending P. aeruginosa against H₂O₂ challenge. In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. However, when each was individually over-expressed in P. aeruginosa no overt H₂O₂-protective phenotype was observed. It was shown that this was due to a masking effect of the P. aeruginosa catalase, KatA; in a katA mutant, H₂O₂ challenged strains over-expressing the WrbA and MdaB orthologs grew significantly better than the empty plasmid control. A growth advantage was also observed for H₂O₂ challenged P. putida strains over-expressing P. aeruginosa wrbA, mdaB or katA. Despite not conferring a growth advantage to wild type P. aeruginosa, it is possible that these quinone oxidoreductases defend against H₂O₂ toxicity at lower concentrations.     

Effects of Hydrogen Sulfide on Growth,Antioxidative Capacity,and Ultrastructural Changes in Oilseed Rape Seedlings Under Aluminum Toxicity

The present study evaluates the beneficial effects of the hydrogen sulfide (H₂S) donor, sodium hydrosulfide (0 and 0.3 mM), on the growth of oilseed rape (Brassica napus L. cv. ZS 758) seedlings under aluminum (Al) stress (0, 0.1, and 0.3 mM). Results showed that Al stress decreased the seedling growth by reducing the shoot and root length, biomass, and antioxidant enzymes, which could be illustrated by increased levels of malondialdehyde (MDA), production of hydrogen peroxide (H₂O₂), and accumulation of Al in the shoots. Pretreatment with H₂S reduced MDA and H₂O₂ levels in the leaves and roots of B. napus seedlings. Moreover, activities of antioxidant enzymes (APX, CAT, APX, SOD, POD, and GR) were elevated significantly with the application of H₂S under Al stress. The microscopic examination confirmed that higher levels of Al completely impaired leaf mesophyll and root tip cells. Chloroplasts were spongy shaped with dissolved thylakoid membranes and more starch grains. Root tip cells showed visible symptoms under Al toxicity such as deposition of Al in vacuoles and disruption of whole cell organelles. Under pretreatment with exogenous H₂S, cell structures were improved and presented a clean mesophyll cell and chloroplast possessing well-developed thylakoid membranes as well as fewer starch grains. A number of modifications could be observed in root tip cells, that is, mature mitochondria, long endoplasmic reticulum as well as golgi bodies, under the combined application of H₂S and Al. On the basis of our results, we can conclude that H₂S has a promotive effect which could improve plant survival under Al stress.     

Influence of sulfur-oxidizing bacteria on the budget of sulfate in Yugama crater lake, Kusatsu-Shirane volcano, Japan

Sulfur-oxidizing bacteria, Thiobacillus thiooxidans, werefound in a highly acidic (pH = 11.5) crater lake, Yugama,seasonally flowing streams and soil in the catchment area of thecrater. Thiobacillus ferrooxidans was also found in some ofthe streams but not in the lake itself. The lake water containsaqueous carbon dioxide, hydrogen sulfide, polythionates andelemental sulfur in suspension which are the substrates for thegrowth of the sulfur-oxidizing bacteria as no organic compoundsexcept for the microorganisms themselves were detected. Thebacteria isolated from the Yugama water preferred polythionatesin the following order: S₄O₆ ²> S₅O₆ ²> S₆O₆ ²On the other hand,H₂S was more rapidly consumed by the bacteria thanpolythionates which were followed by elemental sulfur. In thecase of test-tube incubation, the optimum pH of the solution forgrowth of the bacteria was between 1.0 and 1.5, and forcultivation in growth medium plates between 2.5 and 3.5. Thebacteria hardly proliferated at pH 0.5 or below. In accordancewith these characteristics of the bacteria, numbers of thebacteria in the surface Yugama crater lake water were at minimum(< a few cells/mL) in February and at maximum (10⁶ cell/mL)in August. The bacterial activity changed in accordance with thesurface lake water temperature, but not necessarily with thevariations in H₂S and polythionates concentrations of the lakewater. Based on the variation in sulfur isotope ratios of sulfateand experimentally determined rate of oxidation of H₂S in thelake water, the sulfate production rate by the bacteria in thecatchment area and the lake were estimated to 9.5 and 8.4g/m²/day, respectively, during the period from 1988 to 1990when the volcanic activity at Yugama was at minimum. Also stream,hydrothermal, H₂S-oxidated SO² ₄-inputs and outputs byseepage and precipitation have been calculated as 4.1, 32, 0.56,36, and 1.2 ton/day, respectively.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Hydrogen sulfide inhibits the growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> through oxidative damage

Many studies have shown that hydrogen sulfide (H₂S) is both detrimental and beneficial to animals and plants, whereas its effect on bacteria is not fully understood. Here, we report that H₂S, released by sodium hydrosulfide (NaHS), significantly inhibits the growth of Escherichia coli in a dose-dependent manner. Further studies have shown that H₂S treatment stimulates the production of reactive oxygen species (ROS) and decreases glutathione (GSH) levels in E. coli, resulting in lipid peroxidation and DNA damage. H₂S also inhibits the antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) and induces the response of the SoxRS and OxyR regulons in E. coli. Moreover, pretreatment with the antioxidant ascorbic acid (AsA) could effectively prevent H₂S-induced toxicity in E. coli. Taken together, our results indicate that H₂S exhibits an antibacterial effect on E. coli through oxidative damage and suggest a possible application for H₂S in water and food processing.     

Colorless Sulfur Bacteria, Beggiatoa spp. and Thiovulum spp., in O2 and H2S Microgradients

The interactions between colorless sulfur bacteria and the chemical microgradients at the oxygen-sulfide interface were studied in Beggiatoa mats from marine sediments and in Thiovulum veils developing above the sediments. The gradients of O₂, H₂S, and pH were measured by microelectrodes at depth increments of 50 μm. An unstirred boundary layer in the water surrounding the mats and veils prevented microturbulent or convective mixing of O₂ and H₂S. The two substrates reached the bacteria only by molecular diffusion through the boundary layer. The bacteria lived as microaerophiles or anaerobes even under stirred, oxic water. Oxygen and sulfide zones overlapped by 50 μm in the bacterial layers. Both compounds had concentrations in the range of 0 to 10 μmol liter⁻¹ and residence times of 0.1 to 0.6 s in the overlapping zone. The sulfide oxidation was purely biological. Diffusion calculations showed that formation of mats on solid substrates or of veils in the water represented optimal strategies for the bacteria to achieve a stable microenvironment, a high substrate supply, and an efficient competition with chemical sulfide oxidation. The continuous gliding movement of Beggiatoa cells in mats or the flickering motion of Thiovulum cells in veils were important for the availability of both O₂ and H₂S for the individual bacteria.