Reinhard Bachofen

Effects of natural and chemically synthesized furanones on quorum sensing in Chromobacterium violaceum

BackgroundCell to cell signaling systems in Gram-negative bacteria rely on small diffusible molecules such as the N-acylhomoserine lactones (AHL). These compounds are involved in the production of antibiotics, exoenzymes, virulence factors and biofilm formation. They belong to the class of furanone derivatives which are frequently found in nature as pheromones, flavor compounds or secondary metabolites. To obtain more information on the relation between molecular structure and quorum sensing, we tested a variety of natural and chemically synthesized furanones for their ability to interfere with the quorum sensing mechanism using a quantitative bioassay with Chromobacterium violaceum CV026 for antagonistic and agonistic action. We were looking at the following questions:1. Do these compounds affect growth?2) Do these compounds activate the quorum sensing system of C. violaceum CV026?3) Do these compounds inhibit violacein formation induced by the addition of the natural inducer N-hexanoylhomoserine lactone (HHL)?4) Do these compounds enhance violacein formation in presence of HHL?ResultsThe naturally produced N-acylhomoserine lactones showed a strong non-linear concentration dependent influence on violacein production in C. violaceum with a maximum at 3.7*10-8 M with HHL. Apart from the N-acylhomoserine lactones only one furanone (emoxyfurane) was found to simulate N-acylhomoserine lactone activity and induce violacein formation. The most effective substances acting negatively both on growth and quorum sensing were analogs and intermediates in synthesis of the butenolides from Streptomyces antibioticus.ConclusionAs the regulation of many bacterial processes is governed by quorum sensing systems, the finding of natural and synthetic furanones acting as agonists or antagonists suggests an interesting tool to control and handle detrimental AHL induced effects.Some effects are due to general toxicity; others are explained by a competitive interaction for LuxR proteins. For further experiments it is important to be aware of the fact that quorum sensing active compounds have non-linear effects. Inducers can act as inhibitors and inhibitors might be able to activate or enhance the quorum sensing system depending on chemical structure and concentration levels.

Complexing agents from microorganisms.

The majority of extracellular complexing ligands produced by microorganisms are summarized as being of low molecular mass (<10,000 daltons) and are usually released as part of metal detoxification processes. These exudates appear to exhibit strong metal-binding characteristics, often reducing metal toxicity. Under certain conditions microbes produce metal-specific compounds of low molecular mass called siderophores; although these are normally specific for iron they also have relatively high affinities for radionuclides such as Pu and facilitate their uptake into cells. The occurrence of specific actinide complexing agents has been recorded. The breakdown of lignins and cellulosic material produces large macromolecular compounds called humates. These contain multiligand sites and display a wide range of complexing abilities. They form both soluble and insoluble complexes with toxic elements with various results. Humates also considerably influence adsorption of metals to substrate surfaces and at high pH may compete with OH-ions for metal binding. As well as with extracellular ligands, metals can interact directly with microorganisms by accumulation in subcellular compartments or by adsorption on bacterial surfaces.

Microbial microstratification, inorganic carbon photoassimilation and dark carbon fixation at the chemocline of the meromictic Lake Cadagno (Switzerland) and its relevance to the food web

Abstract: The microstratification of the microbial community at the chemocline of Lake Cadagno and the associated inorganic carbon fixation activity was studied by fine layer sampling. A deep chlorophyll maximum caused by diatoms overlying Cryptomonas was found at the upper edge of the chemocline. A high population density of phototrophic sulphur bacteria, mainly Amoebobacter cf. purpureus, occurred closely below the oxic-anoxic boundary. Despite the small fraction of total lake volume represented by the chemocline, half of the total carbon photoassimilation of the lake occurred within the chemocline with approximately equal contributions by oxygenic and anoxygenic phototrophs. Rates of dark carbon fixation in the chemocline were even higher than rates of photoassimilation, especially at the depths where oxygen and sulphide coexisted during part of the day. These results indicate a substantial contribution by chemolithotrophic organisms to the carbon cycle in Lake Cadagno. Analysis of stable carbon isotopes suggests that zooplankton may obtain as much as half of its carbon at the chemocline, indicating a strong link between production in anoxic waters and the food web in the oxic part of the lake.

Heavy metal conservation in Lake Cadagno sediments: Historical records of anthropogenic emissions in a meromictic alpine lake

Abstract Currently, pollution by heavy metals is one of our most serious environmental problems. Metals such as lead and cadmium enter the biosphere through biogenic and anthropogenic emissions and have been steadily accumulating in soils and sediments. We report on a remote mountain lake in the Swiss Alps where the unusual conditions present have led to the accumulation of several heavy metals in the sediments. The sulfide rich environment contains precipitates of cadmium, lead and zinc. The anoxic conditions and prevalence of sulfate reducing bacteria in the water column have prevented the remobilisation of the metals leaving them trapped in the sediments. This has resulted in the formation of surprisingly “stable” metal profiles which have been correlated to levels of human and industrial activity and provide the means of reconstructing a record of human impact and pollution over the past 50 to 100 years. We suggest that such metals in sediment profiles can also be used as chronological markers in cases where cost or sampling difficulties preclude the use of radiological 137 Cs and 210 Pb dating. Stable metal profiles are valuable sources of environmental data, they reveal pertinent information on the atmospheric transport of contaminants and are relatively simple to analyse.

Biohydrometallurgical processing of solids: a patent review

Autotrophic as well as heterotrophic bacteria and fungi play an important role for the industrial recovery of metals from low-grade ore or, in general, from low-grade mineral resources. The same inorganic bacterial pathways that are responsible for huge and expensive corrosion problems can be used for economical biohydrometallurgical applications. Metals and metalloids can be microbially transformed by oxidation, reduction, alkylation, dealkylation, solubilization, and␣precipitation mechanisms. Biohydrometallurgy, a branch of classical metallurgy, is not as widely publicized as other areas of metallurgy, e.g. pyrometallurgical or hydrometallurgical processes. Since 1990, approximately 15 international patent applications concerning biohydrometallurgical techniques have been claimed under the Patent Cooperation Treaty in contrast to a large number of patents concerning pyro- and hydrometallurgical techniques. Nevertheless, it is a very important field of investigation, especially for the future when aspects of a sustainable development have to be considered. New processes to support this development are applicable, at least on a laboratory scale. Bacterial leaching processes for the recovery of metals from solid residues are applied for low-grade ore, and, more recently, for fly ash, galvanic sludges, or, in general, for industrial wastes. It is possible to recover leached metals and to recycle them in metal-manufacturing industries. In addition, by removing the metals from residues, the environmental quality is improved, and the material can be re-used for construction purposes.

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A Km of 15±1.3 μM CrO42− and a Vmax of 420±50 μmol min−1 mg protein−1 was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO42− and 100±9.6 μmol CrO42− min−1 mg protein−1 for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203.

Seasonal and spatial community dynamics in the meromictic Lake Cadagno.

Abstract. The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8–536) and another specific for the family Chromatiaceae (E. coli positions 8–1005). The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE. The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns. For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae. This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer. All dominant bands were excised and sequenced. Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year. In the chemocline, Chromatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter. This change was confirmed by fluorescent in situ hybridization.

The meromictic alpine Lake Cadagno: Orographical and biogeochemical description

Abstract: Lake Cadagno is a 21 m deep alpine meromictic lake situated at an altitude of 1921 m in the Piora valley in the southern part of central Switzerland. The bedrock of the valley containing dolomite and gypsum determines the chemistry of the water. The lake basin was created by glacial erosion and originally dammed by a glacial moraine. The water body is structured in 3 distinct layers, the oxic mixolimnion, the anoxic monimolimnion and a narrow chemocline in between. The water masses of the lake are stabilized by density differences of salt-rich water which is constantly supplied by subaquatic springs to the monimolimnion. In contrast the mixolimnion is fed by electrolyte-poor surface water. Sulfate, hydrogen carbonate, calcium and magnesium are the dominant ionic species. In the monimolimnion sulfide concentrations of more than 1 mM are found. The chemocline at a depth of 10 to 13 m is characterized by steep chemical and physical gradients. It contains dense populations of up to 105 cells/mL of phototrophic sulfur bacteria consisting of predominantly Chromatium okenii, C. minus and Amoebobacter purpureus. The lake has proven to be an excellent model system for studies of the role of planktonic bacteria which dominate the sulfur cycle.

Characterisation of reaction centers and their phospholipids from Rhodospirillum rubrum

1. Reaction centers from Rhodospirillum rubrum have been extracted with the zwitterionic detergent lauryl dimethyl amine oxide. Subsequent purification has been achieved by gel filtration and ion-exchange chromatography. The pure reaction centers are composed of three protein subunits (L, M, H), bacteriocholorophyll and bacteriopheophytin in the ratio 2 : 1 and phospholipids. 2. The phospholipid composition has been found to be similar to that of whole chromatophore membrane, except that diphosphatidyl glycerol is present in higher amount in the isolated complex. When the detergent treatment of the chromatophore membrane is done in the presence of NaCl, a lower phospholipid content in isolated reaction centers has been found together with a lower stability in the association among the protein subunits. In this complex, the largest subunit H is easily split off and a LM complex is obtained. It is concluded that the phospholipids play an important role in the stability of reaction center complexes.

Growth stimulation of sulfur oxidizing bacteria for optimization of metal leaching efficiency of fly ash from municipal solid waste incineration

Sulfur oxidizing bacteria were used to leach metals bound in municipal solid waste incineration fly ash. Pure or mixed cultures of Thiobacilli with or without addition of anoxic sewage sludge were incubated with various amounts of fly ash for several weeks. Co-cultures with Thiobacilli and sewage sludge grew faster than pure cultures of T. thiooxidans or cultures with only sewage sludge as inoculum. The addition of sewage sludge increased the rate of growth of sulfur oxidizing bacteria and simultaneously enhanced the acidification of the suspension. As a result, the time for fly ash processing was reduced by 50%. Thiobacilli were able to grow in the presence of up to 8% (w/v) fly ash. Depending on the amounts of fly ash added, the final pH, and the concentration of sulfuric acid formed, different amounts of metals were leached: over 80% for Cd, Cu, and Zn, 60% for Al and approx. 30% for Fe and Ni (totally, approx. 50% of the heavy metals present in the fly ash) were extracted.