The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from Methanobrevibacter ruminantium

Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C₁ unit carrier where N⁵‐formyl‐tetrahydromethanopterin is converted to methenyl‐tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N‐terminal domain of six α‐helices encompassing a series of four β‐sheets and a predominantly anti‐parallel β–sheet at the C‐terminus flanked on one side by α‐helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation. Proteins 2013; 81:2064–2070. © 2013 Wiley Periodicals, Inc.     

Biostimulation induces syntrophic interactions that impact C,S and N cycling in a sediment microbial community

Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO₂ during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO₂ production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO₂ fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated.     

Isolation and characterization of alkaliphilic, chemolithoautotrophic, sulphur-oxidizing bacteria

Alkaliphilic sulphur-oxidizing bacteria were isolated from samples from alkaline environments including soda soil and soda lakes. Two isolates, currently known as strains AL 2 and AL 3, were characterized. They grew over a pH range 8.0–10.4 with an optimum at 9.5–9.8. Both strains could oxidize thiosulphate, sulphide, polysulphide, elemental sulphur and tetrathionate. Strain AL 3 more actively oxidized thiosulphate and sulphide, while isolate AL 2 had higher activity with elemental sulphur and tetrathionate. Isolate AL 2 was also able to oxidize trithionate. The pH optimum for thiosulphate and sulphide oxidation was between 9–10. Some activity remained at pH 11, but was negligible at pH 7. Metabolism of tetrathionate by isolate AL 2 involved initial anaerobic hydrolysis to form sulphur, thiosulphate and sulphate in a sequence similar to that in other colourless sulphur-oxidizing bacteria. Sulphate was produced by both strains. During batch growth on thiosulphate, elemental sulphur and sulphite transiently accumulated in cultures of isolates AL 2 and AL 3, respectively. At lower pH values, both strains accumulated sulphur during sulphide and thiosulphate oxidation. Both strains contained ribulose bisphosphate carboxylase. Thiosulphate oxidation in isolate AL 3 appeared to be sodium ion-dependent. Isolate AL 2 differed from AL 3 by its high GC mol % value (65.5 and 49.5, respectively), sulphur deposition in its periplasm, the absence of carboxysomes, lower sulphur-oxidizing capacity, growth kinetics (lower growth rate and higher growth yield) and cytochrome composition.     

Coupling Nutrient Uptake and Energy Flow in Headwater Streams

Nutrient cycling and energy flow in ecosystems are tightly linked through the metabolic processes of organisms. Greater uptake of inorganic nutrients is expected to be associated with higher rates of metabolism [gross primary production (GPP) and respiration (R)], due to assimilatory demand of both autotrophs and heterotrophs. However, relationships between uptake and metabolism should vary with the relative contribution of autochthonous and allochthonous sources of organic matter. To investigate the relationship between metabolism and nutrient uptake, we used whole-stream and benthic chamber methods to measure rates of nitrate–nitrogen (NO₃–N) uptake and metabolism in four headwater streams chosen to span a range of light availability and therefore differing rates of GPP and contributions of autochthonous carbon. We coupled whole-stream metabolism with measures of NO₃–N uptake conducted repeatedly over the same stream reach during both day and night, as well as incubating benthic sediments under both light and dark conditions. NO₃–N uptake was generally greater in daylight compared to dark conditions, and although day-night differences in whole-stream uptake were not significant, light–dark differences in benthic chambers were significant at three of the four sites. Estimates of N demand indicated that assimilation by photoautotrophs could account for the majority of NO₃–N uptake at the two sites with relatively open canopies. Contrary to expectations, photoautotrophs contributed substantially to NO₃–N uptake even at the two closed-canopy sites, which had low values of GPP/R and relied heavily on allochthonous carbon to fuel R.     

Isolation of a strain of Bacillus schlegelii from geothermally heated antarctic soil

Abstract A bacterium capable of growth from 59 to 72° C was isolated from geothermal soil collected from Mount Erebus, Ross Island, Antarctica. The isolate was enriched in medium containing thiosulphate and bicarbonate. Subsequently the organism was found also to be capable of heterotrophic growth and autotrophic growth in the presence of hydrogen and carbon dioxide. In a comparison with Bacillus schlegelii and Bacillus tusciae the isolate most closely resembled B. schlegelii . This conclusion was supported by the finding that B. schlegelii is also capable of autotrophic growth using thiosulphate. The new isolate had a characteristic subunit layer on the cell wall which is typical of B. schlegelii .     

A novel plant-associated thermotolerant alkaliphilic methylotroph of the genusParacoccus

Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium. Cells are cocci or short rods. The strain does not require vitamins. Optimum growth in a medium with methanol occurs at 38–42°C at pH 8.0–9.2. The doubling time is 12 h. In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO₃, and in an atmosphere of H₂ + CO₂ + O₂. Methanol and methylamine are oxidized by the respective dehydrogenases to CO₂ via formaldehyde and formate, respectively. The CO₂ produced is assimilated via the ribulose bisphosphate pathway. Fatty acids are dominated by cyclopropanoic (58–61%), palmitic (24–26%), and octadecanoic (8–9%) acids. The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The major ubiquinone is Q₁₀. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The culture liquid exhibits cytokinin activity. The G+C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature T_m). Strain GB shows a moderate degree of DNA-DNA homology (<40%) with the type representatives of the genusParacoccus. Based on the data obtained, the bacterium was classified as a new species of this genus, namedP. kondratievae.     

Floridosides in unicellular hot spring algae

Cyanidium caldarium strains RK-1, KS-1 and 001 are probably obligate autotrophs, while Chroococcidiopsis sp. strains M-8 and 002 (formerly named Cyanidium caldarium) are facultative autotrophs. The Cyanidium strains contain floridoside (2-O-glycerol-α-d-galactopyranoside) and a small amount of iso-floridoside (1-O-glycerol-α-d-galactopyranoside), both of which are known to be distributed in Rhodophyta. The Chroococcidiopsis strains also contain floridoside, but no iso-floridoside, under various culture conditions. These results indicate that Cyanidium is clearly distinguishable from Chroococcidiopsis in iso-floridoside content and nutritional properties, and suggest that all strains tested may be closely related to Rhodophyta.     

Isolation and characterization of sulfur-utilizing denitrifiers from the sulfur-oxidizing denitrification process

Of 14 potential sulfur-oxidizing strains, Pseudomonas sp. B21 and Agrobacterium sp. B19 were considered as denitrifiers. Under aerobic conditions, with S⁰ as electron donor, maximum cell growth rates were 0.022 (B21) and 0.043 h^–1 (B19). Both grew optimally at pH 7.5 and 28 °C. When NO₃-N was increased from 10 to 200 mg l^–1 the efficiency of nitrate removal of each strain gradually decreased, from 60 to 40%. Addition of suitable organic compounds (C/N < 4.2) increased the nitrate removal efficiencies of both strains, indicating their mixotrophic characters.     

Multivariate analyses of salt stress and metabolite sensing in auto- and heterotroph Chenopodium cell suspensions

Global warming increases plant salt stress via evaporation after irrigation, but how plant cells sense salt stress remains unknown. Here, we searched for correlation-based targets of salt stress sensing in Chenopodium rubrum cell suspension cultures. We proposed a linkage between the sensing of salt stress and the sensing of distinct metabolites. Consequently, we analysed various extracellular pH signals in autotroph and heterotroph cell suspensions. Our search included signals after 52 treatments: salt and osmotic stress, ion channel inhibitors (amiloride, quinidine), salt-sensing modulators (proline), amino acids, carboxylic acids and regulators (salicylic acid, 2,4-dichlorphenoxyacetic acid). Multivariate analyses revealed hirarchical clusters of signals and five principal components of extracellular proton flux. The principal component correlated with salt stress was an antagonism of γ-aminobutyric and salicylic acid, confirming involvement of acid-sensing ion channels (ASICs) in salt stress sensing. Proline, short non-substituted mono-carboxylic acids (C2-C6), lactic acid and amiloride characterised the four uncorrelated principal components of proton flux. The proline-associated principal component included an antagonism of 2,4-dichlorphenoxyacetic acid and a set of amino acids (hydrophobic, polar, acidic, basic). The five principal components captured 100% of variance of extracellular proton flux. Thus, a bias-free, functional high-throughput screening was established to extract new clusters of response elements and potential signalling pathways, and to serve as a core for quantitative meta-analysis in plant biology. The eigenvectors reorient research, associating proline with development instead of salt stress, and the proof of existence of multiple components of proton flux can help to resolve controversy about the acid growth theory.     

Effects of a larval mosquito biopesticide and Culex larvae on a freshwater nanophytoplankton (Selenastrum capricornatum) under axenic conditions

The effects of microbial biopesticides used for mosquito control on autotrophic microorganisms such as nanophytoplankton are equivocal. We examined impacts of mosquito biopesticides and mosquito larvae on primary producers in two independent experiments. In the first experiment, we examined the effects of a commonly used microbial biopesticide formulation (VectoMax^® CG) on a unicellular microalga, Selenastrum capricornatum Printz, under axenic laboratory conditions. The biopesticide treatments included two concentrations (0.008 and 0.016 g liter^?1) of VectoMax^® CG and two controls (one untreated and another with autoclaved 0.016 g VectoMax^® CG liter^?1) in replicated axenic experimental microcosms. Spectrophotometric analysis of chlorophyll a (proxy for algal biomass) and direct enumeration of algal cells following the treatments revealed no significant effects of the microbial biopesticide on algal population growth during the four‐week study. In the second experiment, we tested the effects of different densities of Culex larvae on the population of S. capricornatum. Effects of mosquito larvae feeding on S. capricornatum were significant with a curvilinear relationship between larval density and algal abundance in the water column. Together, these studies demonstrated a lack of direct cytological/toxicological effects of Bacillus‐based microbial pesticides on freshwater primary production and support the hypothesis that the reduction in algal primary production previously reported when Bti products were applied to aquatic environments was likely independent of the Bacillus‐based larvicidal toxins. Instead, it was likely mediated by microbial interactions in the water column and the trophic cascade effects that resulted from the removal of larval mosquitoes. These studies suggest that mosquito larvae independent of pesticide application can influence primary production. Our method of evaluating biopesticides against small photoautotrophs can be very useful for studying the unintended effects on autotrophic microorganisms of other pesticides, including herbicides and pesticides applied to aquatic environments.