Improved screening of biochar compounds for potential toxic activity with microbial biosensors

Biochar is a carbon rich product destined for agricultural use, which can be produced using an array of feedstock and pyrolysis conditions. As such, the resultant biochar product can exhibit characteristics that result in either beneficial or detrimental environmental effects. We set out to establish what the environmental hazards might be for a range of softwood biochars. To facilitate this we conducted biochar headspace analysis, plant germination and bacterial biosensor assays. Our headspace analysis indicated the presence of aldehyde and ester based compounds, which were affected by pyrolysis conditions and in some cases contained compounds that were hazardous to human and environmental health. Germination assays, utilising barley, buckwheat, white clover and oil seed rape, showed that the plants responded differently to the same biochar samples and that in some instances, where germination was unaffected, there were visible physiological effects on seedlings. Finally, we screened water extracts of the biochars under buffered pH, for the presence of potentially toxic elements and compounds using bioluminescence-based bacterial biosensors. Bioluminescence inhibiting compounds were present in extracts prepared from biochar samples pyrolysed at 500 °C under conditions with inadequate control of biochar-volatile interaction. The findings were taken as an indication that bacterial biosensors could be used as a rapid method to screen anomalous biochar products for potential ecotoxicity hazard assessment

Genetic and molecular characterization of multicomponent resistance of Pseudomonas against allicin

The common foodstuff garlic produces the potent antibiotic defense substance allicin after tissue damage. Allicin is a redox toxin that oxidizes glutathione and cellular proteins and makes garlic a highly hostile environment for non-adapted microbes. Genomic clones from a highly allicin-resistant Pseudomonas fluorescens (PfAR-1), which was isolated from garlic, conferred allicin resistance to Pseudomonas syringae and even to Escherichia coli. Resistance-conferring genes had redox-related functions and were on core fragments from three similar genomic islands identified by sequencing and in silico analysis. Transposon mutagenesis and overexpression analyses revealed the contribution of individual candidate genes to allicin resistance. Taken together, our data define a multicomponent resistance mechanism against allicin in PfAR-1, achieved through horizontal gene transfer

Two Gears of Pumping by the Sodium Pump

The kinetics of the phosphorylation and subsequent conformational change of Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na+)3 state. On mixing with ATP in the presence of Mg2+, a fluorescence increase occurred, due to enzyme conversion into the E2P state. The fluorescence change accelerated with increasing ATP concentration until a saturating limit in the hundreds of micromolar range. The amplitude of the fluorescence change (ΔF/F0) increased to 0.98 at 50 μM ATP. ΔF/F0 then decreased to 0.82 at 500 μM. The decrease was attributed to an ATP-induced allosteric acceleration of the dephosphorylation reaction. The ATP concentration dependence of the time course and the amplitude of the fluorescence change could not be explained by either a one-site monomeric enzyme model or by a two-pool model. All of the data could be explained by an (αβ)2 dimeric model, in which the enzyme cycles at a low rate with ATP hydrolysis by one α-subunit or at a high rate with ATP hydrolysis by both α-subunits. Thus, we propose a two-gear bicyclic model to replace the classical monomeric Albers-Post model for kidney Na+,K+-ATPase

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid

The Na+/K+ pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na+ from the cell and import 2K+ per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na+/K+ pump that represent the kinetics of extracellular Na+ binding/release and Na+ occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na+ from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site