Aspects of bioavailability of mercury for methylation in pure cultures of Desulfobulbus propionicus (1pr3)

We have previously hypothesized that sulfide inhibits Hg methylation by decreasing its bioavailability to sulfate-reducing bacteria (SRB), the important methylators of Hg in natural sediments. With a view to designing a bioassay to test this hypothesis, we investigated a number of aspects of Hg methylation by the SRB Desulfobulbus propionicus, including (i) the relationship between cell density and methylmercury (MeHg) production, (ii) the time course of Hg methylation relative to growth stage, (iii) changes in the bioavailability of an added inorganic Hg (Hg(I)) spike over time, and (iv) the dependence of methylation on the concentration of dissolved Hg(I) present in the culture. We then tested the effect of sulfide on MeHg production by this microorganism. These experiments demonstrated that under conditions of equal bioavailability, per-cell MeHg production was constant through log-phase culture growth. However, the methylation rate of a new Hg spike dramatically decreased after the first 5 h. This result was seen whether methylation rate was expressed as a fraction of the total added Hg or the filtered Hg(I) concentration, which suggests that Hg bioavailability decreased through both changes in Hg complexation and formation of solid phases. At low sulfide concentration, MeHg production was linearly related to the concentration of filtered Hg(I). The methylation of filtered Hg(I) decreased about fourfold as sulfide concentration was increased from 10(-6) to 10(-3) M. This decline is consistent with a decrease in the bioavailability of Hg(I), possibly due to a decline in the dissolved neutral complex, HgS(0).     

Complete genome sequence of Desulfobulbus propionicus type strain (1pr3)

Desulfobulbus propionicus Widdel 1981 is the type species of the genus Desulfobulbus, which belongs to the family Desulfobulbaceae. The species is of interest because of its great implication in the sulfur cycle in aquatic sediments, its large substrate spectrum and a broad versatility in using various fermentation pathways. The species was the first example of a pure culture known to disproportionate elemental sulfur to sulfate and sulfide. This is the first completed genome sequence of a member of the genus Desulfobulbus and the third published genome sequence from a member of the family Desulfobulbaceae. The 3,851,869 bp long genome with its 3,351 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Electron Transfer by Desulfobulbus propionicus to Fe(III) and Graphite Electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Electron transfer by Desulfobulbus propionicus to Fe(III) and graphite electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

HCO(3) Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO(3) fixation by these Thiothrix tufts was determined to be 14.0 +/- 5.4 nmol of HCO(3) per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO(3) fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, alpha-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO(3) fixation, 5 to 10 mM malate inhibited HCO(3) fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO(3) at rates as high as 29.9 +/- 2.8 nmol of HCO(3) per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO(3) fixation.     

Nutritional and Other Aspects of Fruit-body Production in Pure Cultures of Collybia velutipes (Curt.) Fr.

Fruit-body production of Collybia velutipes on a synthetic mediumconsisting of minerals, asparagine, sucrose, and vitamin B₁has been studied. Compared with others tested this medium favouredfructification rather than mycelium growth. Primordia aroseon culturea between pH 5•2 and 7•2 approximately.Relatively low initial concentration of asparagine, high initialconcentration of sucrose, and increases in volume (without essentialincrease of the air interface) of media resulted in enhanceddry weights of sporophore crops. These factors variously affectedmycelium yields and the times of primordium production. Detailsof growth, sporophore production, maximum yield, and changesin the medium are given.     

Methods for Measuring Specific Rates of Mercury Methylation and Degradation and Their Use in Determining Factors Controlling Net Rates of Mercury Methylation

A method was developed to estimate specific rates of demethylation of methyl mercury in aquatic samples by measuring the volatile ¹⁴C end products of ¹⁴CH₃HgI demethylation. This method was used in conjunction with a ²⁰³Hg²⁺ radiochemical method which determines specific rates of mercury methylation. Together, these methods enabled us to examine some factors controlling the net rate of mercury methylation. The methodologies were field tested, using lake sediment samples from a recently flooded reservoir in the Southern Indian Lake system which had developed a mercury contamination problem in fish. Ratios of the specific rates of methylation/demethylation were calculated. The highest ratios of methylation/demethylation were calculated. The highest ratios of methylation/demethylation occurred in the flooded shorelines of Southern Indian Lake. These results provide an explanation for the observed increases in the methyl mercury concentrations in fish after flooding.     

Identification of Gibberellins A(1), A(3), and Iso-A(3) in Cultures of Azospirillum lipoferum

Gibberellins A₁, A₃, and iso-A₃ were identified from aseptic cultures of Azospirillum lipoferum strain op 33 by capillary gas chromatography-mass spectrometry (GC-MS) and GC-MS-selected ion monitoring. There were 20 to 40 picograms (in GA₃ equivalents, estimated from bioassay) of gibberellins A₁ and A₃ per milliliter of cell culture (containing 10⁹ cells).     

Methylation of Mercury in Earthworms and the Effect of Mercury on the Associated Bacterial Communities

Methylmercury compounds are very toxic for most organisms. Here, we investigated the potential of earthworms to methylate inorganic-Hg. We hypothesized that the anaerobic and nutrient-rich conditions in the digestive tracts of earthworm''s promote the methylation of Hg through the action of their gut bacteria. Earthworms were either grown in sterile soils treated with an inorganic (HgCl₂) or organic (CH₃HgCl) Hg source, or were left untreated. After 30 days of incubation, the total-Hg and methyl-Hg concentrations in the soils, earthworms, and their casts were analyzed. The impact of Hg on the bacterial community compositions in earthworms was also studied. Tissue concentrations of methyl-Hg in earthworms grown in soils treated with inorganic-Hg were about six times higher than in earthworms grown in soils without Hg. Concentrations of methyl-Hg in the soils and earthworm casts remained at significantly lower levels suggesting that Hg was mainly methylated in the earthworms. Bacterial communities in earthworms were mostly affected by methyl-Hg treatment. Terminal-restriction fragments (T-RFs) affiliated to Firmicutes were sensitive to inorganic and methyl-Hg, whereas T-RFs related to Betaproteobacteria were tolerant to the Hg treatments. Sulphate-reducing bacteria were detected in earthworms but not in soils.     

Improved Enrichment and Isolation Procedures for Obtaining Pure Cultures of Beggiatoa

Scoring agar surfaces with alginate swabs before placing washed filaments of Beggiatoa on the agar has greatly increased the rate at which single filaments move from contaminated areas. Numerous morphological types of pure cultures have been grown in organic media supplemented with either catalase or reducing agents. Aerated sewage was used as the enrichment source.     

Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B₁₂ in some and perhaps many incomplete-oxidizing SRB strains.     

Effect of Aflatoxin B(1) on Cell Cultures.

Gabliks, J. (Massachusetts Institute of Technology, Cambridge), W. Schaeffer, L. Friedman, and G. Wogan. Effect of aflatoxin B(1) on cell cultures. J. Bacteriol. 90:720-723. 1965.-Aflatoxin B(1), a metabolite of the mold Aspergillus flavus, is toxic to cell cultures. The toxic effect is evidenced by an inhibition of growth followed by progressive granulation, rounding, and finally sloughing of the cells from the glass. In studies with embryonated eggs, duck embryos were found to be four to five times more susceptible than chick embryos. In studies on Chang liver cultures, there were decreases in cell number, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) per culture with increasing aflatoxin B(1) concentrations. Since the cell number decreased and the protein, RNA, and DNA content per cell increased with increasing concentrations of aflatoxin, enlarged cells were suggested. These data are consistent with in vivo data of other workers who have found hypertrophic cells with enlarged nuclei in histological studies on the tissues of rats and ducklings fed toxic peanut meal.     

Mercury Methylation by Desulfovibrio desulfuricans ND132 in the Presence of Polysulfides

The extracellular speciation of mercury may control bacterial uptake and methylation. Mercury-polysulfide complexes have recently been shown to be prevalent in sulfidic waters containing zero-valent sulfur. Despite substantial increases in total dissolved mercury concentration, methylation rates in cultures of Desulfovibrio desulfuricans ND132 equilibrated with cinnabar did not increase in the presence of polysulfides, as expected due to the large size and charged nature of most of the complexes. In natural waters not at saturation with cinnabar, mercury-polysulfide complexes would be expected to shift the speciation of mercury from HgS⁰_(aq) toward charged complexes, thereby decreasing methylation rates.     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.