Environmental stimuli are sensed through transient [Ca2+]i elevations by M. loti To further validate the experimental system, abiotic stimuli which are known to trigger [Ca2+]i changes in both plants [23] and cyanobacteria [18, 19] were applied to apoaequorin-expressing M. loti cells. A mechanical perturbation, simulated by the injection of isoosmotic cell culture medium, resulted in a rapid Ca2+ transient increase (1.08 ± 0.24 μM) that decayed within 30 sec (Fig. 1A). This Ca2+ trace, which is frequently referred to as a “”touch response”", is often observed after the

hand-operated injection of any stimulus [24]. A similar Ca2+ response characterized by an enhanced Ca2+ peak of 2.14 ± 0.46 μM was triggered by a click here simple injection of air into the cell suspension with a needle (Fig. 1A). Figure 1 Ca 2+ measurements in M. loti

cells stimulated with different physico-chemical signals. Bacteria were challenged (arrow) with: A, mechanical perturbation, represented by injection of an equal volume of culture medium (black trace) or 10 volumes of air (grey trace); B, cold shock, given by 3 volumes of ice-cold culture medium (black BMN 673 research buy trace); control cells were stimulated with 3 volumes of growth medium kept at room temperature (grey trace); C, hypoosmotic stress, given by injection of 3 volumes of distilled water (black trace); salinity stress, represented by 200 mM NaCl (grey trace); D, different external Ca2+ concentrations. These and the following traces have been chosen Interleukin-2 receptor to best represent the average results of at least three independent experiments. Cold and hypoosmotic shocks, caused by supplying three volumes of ice-cold medium and distilled water, respectively, induced Ca2+ traces with distinct kinetics, e.g. different height of the Ca2+ peak (1.36 ± 0.13 μM and 4.41 ± 0.51 μM, respectively) and rate

of dissipation of the Ca2+ signal (Fig. 1B and 1C). As a control, cells were stimulated with three volumes of growth medium at room temperature, (Fig. 1B) resulting in a Ca2+ trace superimposable on that of the touch response (Fig. 1A). These findings eliminate the possible effect of bacterial dilution on changes in Ca2+ homeostasis. Challenge of M. loti with a salinity stress, which has recently been shown to affect symbiosis-related events in Rhizobium tropici [25], resulted in a [Ca2+]i elevation of large amplitude (3.36 ± 0.24 μM) and a specific signature (Fig. 1C). Variations in the extracellular Ca2+ concentration determined the induction of transient Ca2+ elevations whose magnitude was dependent on the level of external Ca2+. After a rapidly induced increase in [Ca2+]i, the basal Ca2+ level was gradually restored with all the applied external Ca2+ concentrations (Fig. 1D), confirming a tight internal homeostatic Ca2+ control, as previously shown for other bacteria [14, 18]. All the above results indicate that aequorin-expressing M.