Probing the mechanical properties of magnetosome chains in living magnetotactic bacteria.

The mechanical properties of cytoskeletal networks are intimately involved in determining how forces and cellular processes are generated, directed, and transmitted in living cells. However, determining the mechanical properties of subcellular molecular complexes in vivo has proven to be difficult. Here, we combine in vivo measurements by optical microscopy, X-ray diffraction, and transmission electron microscopy with theoretical modeling to decipher the mechanical properties of the magnetosome chain system encountered in magnetotactic bacteria. We exploit the magnetic properties of the endogenous intracellular nanoparticles to apply a force on the filament-connector pair involved in the backbone formation and stabilization. We show that the magnetosome chain can be broken by the application of external field strength higher than 30 mT and suggest that this originates from the rupture of the magnetosome connector MamJ. In addition, we calculate that the biological determinants can withstand in vivo a force of 25 pN. This quantitative understanding provides insights for the design of functional materials such as actuators and sensors using cellular components.

Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae).

Humivorous scarab beetle larvae can thrive exclusively on soil organic matter. Feeding experiments have revealed that the larva of Pachnoda ephippiata mineralizes all major humus components except the polyphenolic fraction. High proteolytic activity in the alkaline midgut fluid and an enormous ammonia production during gut passage suggested that peptidic soil components are an important dietary resource for the larva. By comparing acid-hydrolyzable amino acids in food soil and feces, we showed that a significant fraction of the peptides in soil are removed during gut passage. This agrees well with the high concentrations of free amino acids found the midgut section. Incubation experiments revealed the presence of substantial particle-associated proteolytic activity also in the hindgut, most probably due to microbial activity. High rates of ammonia formation in hindgut homogenates and the conversion of radiolabeled amino acids to acetate and propionate indicated that microbial fermentations of soil peptides play an important role in the hindgut. This was corroborated by viable counts of amino-acid-fermenting bacteria, which formed a substantial fraction of the hindgut microbiota. A complete inventory of organic and inorganic nitrogen species before, during, and after gut passage revealed the formation of nitrite and nitrate in midgut and hindgut, and a substantial nitrogen deficit in the feces, suggesting that part of the ammonia formed by mineralization is subjected to oxidation and subsequent denitrification to N2. Together, the results strongly support the hypothesis that peptidic soil components form a major energy and nutrient source for humivorous insects, supplying the animal with microbial fermentation products and essential amino acids.

Factors controlling the carbon isotope fractionation of tetra- and trichloroethene during reductive dechlorination by Sulfurospirillum ssp. and Desulfitobacterium sp. strain PCE-S.

Carbon stable isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE) was investigated during reductive dechlorination. Growing cells of Sulfurospirillum multivorans, Sulfurospirillum halorespirans, or Desulfitobacterium sp. strain PCE-S, the respective crude extracts and the abiotic reaction with cyanocobalamin (vitamin B(12)) were used. Fractionation of TCE (alphaC=1.0132-1.0187) by S. multivorans was more than one order of magnitude higher than values previously observed for tetrachloroethene (PCE) (alphaC=1.00042-1.0017). Similar differences in fractionation were observed during reductive dehalogenation by the close relative S. halorespirans with alphaC=1.0046-1.032 and alphaC=1.0187-1.0229 for PCE and TCE respectively. TCE carbon isotope fractionation (alphaC=1.0150) by the purified PCE-reductive dehalogenase from S. multivorans was more than one order of magnitude higher than fractionation of PCE (alphaC=1.0017). Carbon isotope fractionation of TCE by Desulfitobacterium sp. strain PCE-S (alphaC=1.0109-1.0122) as well as during the abiotic reaction with cyanocobalamin (alphaC=1.0154) was in a similar range to previously reported values for fractionation by mixed microbial cultures. In contrast with previous results with PCE, no effects due to rate limitations, uptake or transport of the substrate to the reactive site could be observed during TCE dechlorination. Our results show that prior to a mechanistic interpretation of stable isotope fractionation factors it has to be carefully verified how other factors such as uptake or transport affect the isotope fractionation during degradation experiments with microbial cultures.

Anaerobic degradation of increased phenol concentrations in batch assays.

Phenol is a wastewater contaminant depicting an environmental hazard. It can be found in effluents from various industrial processes and becomes even more common as a waste by-product of biomass-based bioenergy concepts. Because of its toxicity to anaerobic microorganisms, it can be recalcitrant during biogas production and anaerobic wastewater treatment. This study tested increased phenol loads (100 to 5000 mg L(-1)) as the sole carbon source in a semi-continuous mesophilic anaerobic adaption experiment using an unadapted microbial community from a standard biogas plant. Phenol was completely degraded at starting concentrations of up to 2000 mg L(-1). At 5000 mg L(-1), complete inhibition of the anaerobic community was observed. Lag times were reduced down to less than a day treating 2000 mg L(-1) after 16 weeks of adaption to gradually increased phenol concentrations. Specific degradation rates increased consecutively up to 7.02 mg gVS (-1) day(-1) at 2000 mg L(-1). This concentration was completely degraded within less than 12 days. The microbial community composition was assessed using 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis. In the bacterial community, no clear shift was visible. Clostridia were with the highest relative abundance of 27 %, the most prominent bacterial class. T-RFs representing Clostridia, Anaerolinaceae, Flavobacteria, and Bacteroidea appeared at similar relative abundance level throughout the experiment. The archaeal community, however, changed from a Methanosarcinales-dominated community (57%) to a community with a nearly even distribution of Methanobacteriales (21%) and Methanosarcinales (34%) with increasing starting phenol concentration.

Effects of different biochars and digestate on N2O fluxes under field conditions.

Field studies that have investigated the effects of char materials on the emission of nitrous oxide (N2O) are still scarce. Therefore, we conducted a field trial with bio- and hydrochars and measured N2O emissions for one whole year. It was hypothesised that the incorporation of chars reduces the emissions of N2O. Chars were produced by pyrolysis and hydrothermal carbonisation (HTC) using either maize silage or wood residues as feedstock. In addition, after production chars were post-treated with digestate in order to accelerate the ageing process of the chars. Chars and digestate were applied to the soil to raise the C content. Emissions of N2O were measured weekly and soil samples for inorganic nitrogen (N) and soil water-content were taken once a month. Additionally, the abundance of functional marker genes from denitrification (nosZ) was determined in October 2012 and in June 2013. The treatment with pure digestate emitted the most N2O compared to the control and char treatments. However, this was significant only in one case. There were no great differences between the char treatments due to high spatial variability and gene abundance of nosZ did not differ between treatments. Overall, emissions of N2O were relatively low. This was attributed to the heterogeneous distribution of the chars and the sandy soils that did not favour the production of N2O. To conclude, the emissions of N2O were mainly influenced by temperature and precipitation and to a minor extent by the type of char and post-treatment.

Inter- and intraspecific comparison of the bacterial assemblages in the hindgut of humivorous scarab beetle larvae (Pachnoda spp.).

The larvae of scarab beetles are model organisms for studying the role of physicochemical gut conditions and intestinal microbiota in symbiotic digestion, particularly of humus. Here, we address the question of whether the enlarged hindgut paunch of Pachnoda ephippiata and Pachnoda marginata, two closely related, but allopatric species, harbors a specific bacterial microbiota. Terminal restriction length fragment polymorphism (T-RFLP) analysis revealed that in both species, the bacterial hindgut community differs strongly from that in the midgut, food soil, and fecal pellets. High intra- and interspecific similarities between the T-RFLP profiles of different larvae indicate the presence of a hindgut-specific microbiota. Nevertheless, we found a clear separation of the two species. A 16S rRNA gene clone library from the hindgut of P. ephippiata identified the major phylogenetic groups as members of the Clostridia, Betaproteobacteria, and Bacteroidetes, followed by Bacillales and Deltaproteobacteria. A comparison with a previously obtained clone library of the same species corroborates both the similarities and the intraspecific variance of the hindgut microbiota.

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin.

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with alphaC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (alphaC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (alphaC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, alphaC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.

Synthesis and characterization of metal binding pseudotripeptides.

Metal complexes with peptide or pseudopeptide type ligands can serve as good model compounds for a deeper understanding of enzymatic catalysis, but ligands with a high selectivity for different transition metal cations are hard to find due to the rather flexible nature of peptides. Since such ligands would be the sine qua non condition for the synthesis of heterodinuclear peptide metal complexes with catalytic activity, the search for small, affine and selective metal chelating sequences is of interest. Using four different amino acids (His, Lys, Asp, Glu) a set of 16 pseudotripeptides of the common structure Bz-AS1-Sar-AS2-NH2 has been synthesized, purified and characterized by mass spectrometry and 1H-NMR. Their ability to form metal complexes has been investigated leading to short motifs capable of selectively binding only one or two transition metal cations with high affinity. As expected, the complexation of transition metal cations by pseudotripeptides is strongly dependent not only on the amino acid composition, but also on the sequence with regard to the stability of the resulting complexes, as well as the selectivity of the ligands towards Cu2+, Co2+, Ni2+, Zn2+ and Mn2+.