In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homolog FtsZ at mid-cell to form a cell division scaffold (the Z ring), followed by recruitment of the other divisome components. The current view of bacterial cell division control starts from the principle of negative checkpoints that prevent incorrect Z-ring positioning. Here we provide evidence of positive control of cell division during sporulation of Streptomyces, via the direct recruitment of FtsZ by the membrane-associated divisome component SsgB. In vitro studies demonstrated that SsgB promotes the polymerization of FtsZ. The interactions are shown in vivo by time-lapse imaging and Fö rster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM), and are corroborated independently via two-hybrid studies. As determined by fluorescence recovery after photobleaching (FRAP), the turnover of FtsZ protofilaments increased strongly at the time of Z-ring formation. The surprising positive control of Z-ring formation by SsgB implies the evolution of an entirely new way of Z-ring control, which may be explained by the absence of a mid-cell reference point in the long multinucleoid hyphae. In turn, the localization of SsgB is mediated through the orthologous SsgA, and premature expression of the latter is sufficient to directly activate multiple Z-ring formation and hyperdivision at early stages of the Streptomyces cell cycle.

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaH degrees '(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic "driving force" for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.

The reduction thermodynamics (Delta H degrees '(rc) and Delta S degrees '(rc)) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E degrees ', which is mainly determined by solvent reorganization effects. Analysis of all the Delta H degrees '(rc) and Delta S degrees '(rc) values available for this protein class indicates that enthalpy-entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.