The spatial organization of the active sites of the bifunctional oligomeric enzyme tryptophan synthase: Cross-linking by a novel method

To achieve specific cross-linking between the active sites of the non-identical subunits tryptophan synthase from E. coli was modified by a novel method. After reaction with bifunctional reagents of the isolated subunits at their active sites, the tetrameric complex was formed and the free ends of the reagent molecules reacted with each other forming a covalent bridge between the subunits. The distance between the amino acid side chains involved in the cross-linking should not exceed approx. 1.8 nm. A distance much shorter than that is unlikely since all attempts to cross-link the active sites with different shorter bifunctional reagents failed. The implications of these results in the mechanism of action of the enzyme are discussed.     

Microbial cycling of iron and sulfur in sediments of acidic and pH-neutral mining lakes in Lusatia (Brandenburg, Germany)

A vast number of lakes developed in the abandoned opencast lignite mines of Lusatia (East Germany) contain acidic waters (mmolSm^–2a^–). Potential Fe(III) reduction measured by the accumulation of Fe(II) during anoxic incubation yielded similar rates in both types of sediments, however, the responses towards the supplementation of Fe(III) and organic carbon were different. Sulfate reduction rates estimated with ³⁵S-radiotracer were much lower in the slightly acidic sediment than in the pH-neutral sediment (156 v.s. 738mmolSO₄ ^2–m^–2a^–1). However, sulfate reduction rates were increased by the addition of organic carbon. Severe limitation of sulfate-reducing bacteria under acidic conditions was also reflected by low most probable numbers (MPN). High MPN of acidophilic iron- and sulfur-oxidizing bacteria in acidic sediments indicated a high reoxidation potential. The results show that potentials for reductive processes are present in acidic sediments and that these are determined mainly by the availability of oxidants and organic matter.     

Members of a readily enriched beta-proteobacterial clade are common in surface waters of a humic lake

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. beta-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments. In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment. Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.     

The hydrogeology of a catchment area and an artificially divided dystrophic lake — consequences for the limnology of Lake Fuchskuhle

The hydrogeology between the catchment area and the divided dystrophic Lake Fuchskuhle with respect to the genesis and the land-water interactions were investigated. Water levels at numerous locations in the catchment area were measured in order to characterize the hydrology. The water balance of the area was calculated based on long term climatic investigations. The geology of the peat was documented at 25 sampling points by cores collected with a peat drill. Chemical parameters including pH, total phosphorus and total nitrogen concentrations, DOC concentration, colour (SAK 436 m^–1) and the UV₂₅₄/DOC ratio in the catchment area and in two compartments (NE and SW compartment) were determined. The chemical fluxes of DOC, nitrogen and phosphorus from the catchment area into one compartment (SW compartment) were determined. During the genesis of the Lake Fuchskuhle area two aquifer systems (local peat aquifer, regional sandy main aquifer) developed. Both aquifers are largely independently with almost no lateral interactions. Two compartments are supplied with water from the local peat aquifer. From the other two compartments, however, water is flowing out into the peat body. During high groundwater inflow into the SW compartment higher concentration of DOC, nitrogen and phosphorus in the SW compartment were detected. The fen can be divided in two parts: in the meso — to eutrophic fen northwest and the mainly meso — to oligotrophic — acid fen in the southeast. The significant differences in parameters such as pH, conductivity and DOC concentration gave a clear picture of the heterogeneity of the two compartments and their dependence on the catchment area with the two aquifers.     

Pressure-induced structural changes of pig heart lactic dehydrogenase

Lactic dehydrogenase from pig heart can be reversibly dissociated at hydrostatic pressures above 1000 bar. The breakdown of the native quaternary structure occurs at lower pressures compared to the isoenzyme from pig, skeletal muscle. As shown by hybridization experiments of the two isoenzymes the final product of dissociation is the homogeneous monomer. Fluorescence emission spectra of the monomeric enzyme at elevated pressure are characterized by a decrease in fluorescence intensity without any red shift, indicating that no significant unfolding occurs upon high-pressure dissociation. The spectral changes are comparable to those observed after acid dissociation. The amount and rate of deactivation depend on pressure and on the conditions of the solvent. The presence of various anions (Cl^?, SO^2?₄. HPO₄^2?) has no effect on the stability of ihe enzyme towards pressure. High-pressure denaturation (as monitored by intrinsic protein fluorescence), and deactivalion (measured immediately after decompression) run parallel; the pressure dependence of their first-order rate constants is characterized by an activation volume ΔV^≠_Dc = ?140 = 10 cm³/mol. As taken from the yield of reconstitution, dissociation, denaturation and deactivation are found to be fully reversible provided the pressure does not exceed a limiting value (p = 1000 bar in Tris. pH 7.6: 24 h incubation at 20°C). After extended incubation beyond the limiting, pressure of 1000 bar. “irreversible high-pressure denaturation” occurs which is accompanied bv partial aggregation after decompression. The coenzyme, NAD⁺ stabilizes the native tetramer shifting the dissociation equilibrium to higher pressures. The overall dissociation-association reaction can be quantitatively described by a consecutive dissociation/unfolding mechanism N?4 M^'?4 M^☆ (where N is the native tetramer. and M^' and M^☆ two different conformations of the monomer). The reaction volume of the dissociation reaction N?4 M^' is found to be ΔV_Diss = ?360 = 30 cm³/mol: as indicated by the pressure dependence of the yield of reconstitution, the reaction volume of the equilibrium M^'?M^XXX is also negative.     

High genetic and physiological diversity of sulfate-reducing bacteria isolated from an oligotrophic lake sediment

The community structure of sulfate-reducing bacteria in littoral and profundal sediments of the oligotrophic Lake Stechlin (Germany) was investigated. A collection of 32 strains was isolated from the highest positive dilutions of most-probable-number series, and their partial 16S rRNA gene sequences and genomic fingerprints based on ERIC (enterobacterial repetitive intergenic consensus)-PCR were analyzed. The strains fell into eight distinct phylogenetic lineages, and the majority (70%) showed a close affiliation to the genus Desulfovibrio. Most of the remaining strains (22%) were related to the gram-positive Sporomusa and Desulfotomaculum groups. A high redundancy of 16S rRNA gene sequences was found within several of the phylogenetic lineages. This low phylogenetic diversity was most pronounced for the subset of strains isolated from oxic sediment layers. ERIC-PCR revealed that most of the strains with identical 16S rRNA gene sequences were genetically different. Since strains with identical 16S rRNA gene sequences but different genomic fingerprints also differed considerably with respect to their physiological capabilities, the high diversity detected in the present work is very likely of ecological relevance. Our results indicate that a high diversity of sulfate-reducing bacterial strains can be recovered from the natural environment using the established cultivation media. Received: 20 April 1998 / Accepted: 12 June 1998     

Desulfosporomusa polytropa gen. nov., sp. nov., a novel sulfate-reducing bacterium from sediments of an oligotrophic lake

Five strains of sulfate-reducing bacteria were isolated from the highest positive dilutions of a most probable number (MPN) series supplemented with lactate and inoculated with sediments from the oligotrophic Lake Stechlin. The isolates were endospore-forming and were motile by means of laterally inserted flagella. They stained Gram-negative and contained b-type cytochromes. CO difference spectra indicated the presence of P582 as a sulfite reductase. Phylogenetic analyses of the 16S rDNA sequences revealed that the isolates were very closely affiliated with the genus Sporomusa. However, sulfate and amorphous Fe(OH)₃, but not sulfite, elemental sulfur, MnO₂, or nitrate were used as terminal electron acceptors. Homoacetogenic growth was found with H₂/CO₂ gas mixture, formate, methanol, ethanol, and methoxylated aromatic compounds. The strains grew autotrophically with H₂ plus CO₂ in the presence or absence of sulfate. Formate, butyrate, several alcohols, organic acids, carbohydrates, some amino acids, choline, and betaine were also utilized as substrates. The growth yield with lactate and sulfate as substrate was 7.0 g dry mass/mol lactate and thus two times higher than in sulfate-free fermenting cultures. All isolates were able to grow in a temperature range of 4–37°C. Physiologically and by the presence of a Gram-negative cell wall, the new isolates resemble known Desulfosporosinus species. However, phylogenetically they are affiliated with the Gram-negative genus Sporomusa belonging to the Selenomonas subgroup of the Firmicutes. Therefore, the new isolates reveal a new phylogenetic lineage of sulfate-reducing bacteria. A new genus and species, Desulfosporomusa polytropa gen. nov., sp. nov. is proposed.Dedicated to Prof. H. G. Schlegel on the occasion of his 80th birthday.