In situ mapping of nitrifiers and anammox bacteria in microbial aggregates by means of confocal resonance Raman microscopy

In the present work the exploration of microbial communities by confocal resonance Raman microscopy (CRRM) is reported. Using the resonance Raman effect of cytochrome c (Cyt c) we were able to record the microbial distribution of nitrifiers and anammox bacteria directly in their natural environment without the need of sample preparation. For this new non-invasive investigation a reference database of bacteria assumed to be found in microbial aggregates obtained from biological wastewater treatment was created. Reference spectra of enriched cultures of the interesting bacteria were taken by means of optical tweezers. Significant spectra were achieved in less than 1 s (excitation wavelength 532 nm, laser power 9 mW) due to the resonance Raman effect. In addition, the impact of different parameters on the reference spectra, such as integration time and culture conditions, was analysed. We successfully demonstrate the grouping of bacteria down to strain level based on the heterogeneity of the resonance Raman spectra of the heme protein Cyt c.     

Depolymerization-driven flow in nematode spermatozoa relates crawling speed to size and shape

Cell crawling is an inherently physical process that includes protrusion of the leading edge, adhesion to the substrate, and advance of the trailing cell body. Research into advance of the cell body has focused on actomyosin contraction, with cytoskeletal disassembly regarded as incidental, rather than causative; however, extracts from nematode spermatozoa, which use Major Sperm Protein rather than actin, provide at least one example where cytoskeletal disassembly apparently generates force in the absence of molecular motors. To test whether depolymerization can explain force production during nematode sperm crawling, we constructed a mathematical model that simultaneously describes the dynamics of both the cytoskeleton and the cytosol. We also performed corresponding experiments using motile Caenorhabditis elegans spermatozoa. Our experiments reveal that crawling speed is an increasing function of both cell size and anterior-posterior elongation. The quantitative, depolymerization-driven model robustly predicts that cell speed should increase with cell size and yields a cytoskeletal disassembly rate that is consistent with previous measurements. Notably, the model requires anisotropic elasticity, with the cell being stiffer along the direction of motion, to accurately reproduce the dependence of speed on elongation. Our simulations also predict that speed should increase with cytoskeletal anisotropy and disassembly rate.     

The influence of altitude on the distribution of subterranean organs and humus components in Vaccinium myrtillus carpets

Abstract. Humus profiles were sampled along an altitudinal gradient in the Macot‐La‐Plagne Forest (France, northern Alps) to investigate variation occurring under carpets of Vaccinium myrtillus present within Picea abies forests. The vertical distribution of subterranean organs of V. myrtillus was compared with (1) that of P. abies roots and other accompanying vegetation and (2) other components of humus profiles, in particular humified organic matter mainly consisting of animal faeces. It was shown that V. myrtillus roots were mostly concentrated in mineral horizons, while P. abies roots and V. myrtillus rhizomes occupied litter horizons. This was interpreted in terms of competition for nutrient capture between P. abies and V. myrtillus. The effects of altitude were (1) a change in the vegetation accompanying V. myrtillus in dense V. myrtillus carpets, bryophytes at the montane level being replaced by forbs at the sub‐alpine level and (2) a decrease in the thickness of ecto‐organic horizons. This was interpreted as a shift from a moder system characterized by recalcitrant litter (moss) processed by an active faunal community (stabilized in the form of animal faeces) to a mor system characterized by low animal abundance but with litter of better quality which is easily leached in the absence of prominent faunal activity.     

Mode of Action of Ponazuril Against Toxoplasma gondii Tachyzoites in Cell Culture

ABSTRACT. Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We investigated the inode of action of ponazuril against developing RH strain T. gondii tachyzoites in African green monkey kidney cells. Host cells were infected with  2.0 × 10⁵  tachyzoites and treated with 5 μg/ml ponazuril. Cultures were fixed and examined by transmission electron microscopy 3 days after treatment. Ponazuril interfered with normal parasite division. This led to the presence of multinucleate schizonts stages. Up to six tachyzoites were observed partially budded from the surface of these schizonts. Large vacuoles developed in these schizonts and they eventually degenerated.     

Solution structure of copper ion-induced molecular aggregates of tyrosine melanin.

Melanin, the ubiquitous biological pigment, provides photoprotection by efficient filtration of light and also by its antioxidant behavior. In solutions of synthetic melanin, both optical and antioxidant behavior are affected by the aggregation states of melanin. We have utilized small-angle x-ray and neutron scattering to determine the molecular dimensions of synthetic tyrosine melanin in its unaggregated state in D(2)O and H(2)O to study the structure of melanin aggregates formed in the presence of copper ions at various copper-to-melanin molar ratios. In the absence of copper ions, or at low copper ion concentrations, tyrosine melanin is present in solution as a sheet-like particle with a mean thickness of 12.5 A and a lateral extent of approximately 54 A. At a copper-to-melanin molar ratio of 0.6, melanin aggregates to form long, rod-like structures with a radius of 32 A. At a higher copper ion concentration, with a copper-to-melanin ratio of 1.0, these rod-like structures further aggregate, forming sheet-like structures with a mean thickness of 51 A. A change in the charge of the ionizable groups induced by the addition of copper ions is proposed to account for part of the aggregation. The data also support a model for the copper-induced aggregation of melanin driven by pi stacking assisted by peripheral Cu(2+) complexation. The relationship between our results and a previous hypothesis for reduced cellular damage from bound-to-melanin redox metal ions is also discussed.     

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.     

The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor

Triosephosphate isomerase (TIM) has a conserved salt bridge 20 A away from both the active site and the dimer interface. In this study, four salt bridge mutants of Trypanosoma brucei brucei TIM were characterized. The folding and stability of the mutants are impaired compared to the wild-type enzyme. This salt bridge is part of a hydrogen bonding network which tethers the C-terminal beta7alpha7beta8alpha8 unit to the bulk of the protein. In the variants D227N, D227A, and R191S, this network is preserved, as can be deduced from the structure of the R191S variant. In the R191A variant, the side chain at position 191 cannot contribute to this network. Also the catalytic power of this variant is most affected.     

Towards a new attenuating compound: a potentiometric, spectrophotometric and NMR equilibrium study on Fe(III), Al(III) and a new tetradentate mixed bisphosphonate-hydroxypyridinonate ligand

Coordination properties toward Fe(III) and Al(III) of a mixed bisphosphonate-hydroxypyridinonate ligand are presented. Potentiometric, spectrophotometric and NMR results allowed to conclude that Fe(III) and Al(III) coordination takes place on the pyridinone moiety. The high steric hindrance prevents the possibility of simultaneous coordination of both groups to the same metal ion. Quantum mechanical calculations confirm this finding allowing to determine the minimal length of the linker necessary for a stable conformation of complexes in which Fe(III) is coordinated both by pyridinone and bisphosphonate groups.