Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Escherichia coli</i>.

<p>Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Escherichia coli</i>.</p

Eyring plots of the GTPase activity and polymerization of FtsZ.

<p>Top row, mesophilic EcFtsZ. Bottom row, thermophilic MjFtsZ. The kinetic rates of GTP hydrolysis, <i>k_cat</i> (A and C), and filament depolymerization, <i>k_depol</i> (B and D), are plotted as a function of temperature. The apparent enthalpies (Δ<i>H</i>^0‡) and entropies (Δ<i>S</i>^0‡) of the transition state were calculated by nonlinear regression using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e006" target="_blank">Eq 4</a> (solid lines), and the best-fit values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t003" target="_blank">Table 3</a>.</p

The morphology of FtsZ polymers was characterized by negative-stain transmission electron microscopy.

<p>Top row, 10 μM EcFtsZ was polymerized at 10, 20 and 30°C (A-C). Bottom row, 10 μM MjFtsZ was polymerized at 40, 60 and 80°C (D–F). The arrowheads in D point to examples of short and curved polymers. The scale bars represent 100 and 500 nm for the top and bottom rows, respectively (black bars).</p

Temperature dependence of the GTP hydrolysis rates of FtsZ.

<p>The effect of temperature and protein concentration on the GTPase activity of FtsZ was examined for mesophilic EcFtsZ (A) and for thermophilic MjFtsZ (B). The catalytic constant <i>k_cat</i> was determined from the slopes of the linear regressions indicated by the solid lines (the substrate was used at saturating concentrations). The critical concentrations C_C-GTPase were obtained from the intersection of the linear regressions with the protein concentration axis. The symbols and error bars are the averages and the standard deviations from triplicate samples. The calculated values of C_C-GTPase and <i>k_cat</i> are presented in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t002" target="_blank">2</a>.</p

Transition state enthalpy, entropy and free energy of FtsZ assembly.

<p>Transition state enthalpy, entropy and free energy of FtsZ assembly.</p

Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Methanocaldococcus jannaschii</i>.

<p>Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Methanocaldococcus jannaschii</i>.</p

Global analysis of the critical concentration using the integrated van′t Hoff equation.

<p>The combined data obtained with the GTP hydrolysis and polymerization assays were fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e008" target="_blank">Eq 6</a> using nonlinear regression (solid lines), for mesophilic EcFtsZ (A) and thermophilic MjFtsZ (B). The fitting coefficients for EcFtsZ are: <i>a</i> = -787.59, <i>b</i> = 38,745 and <i>c</i> = 117.86, and the coefficients obtained for MjFtsZ: <i>a</i> = 776.24, <i>b</i> = -40,253 and <i>c</i> = -110.53. The temperature-dependent parameters Δ<i>H</i>⁰ and Δ<i>S</i>⁰ were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e010" target="_blank">Eq 7</a> (Panels C and D), as well as the temperature-independent heat capacity change Δ<i>C</i>_<i>p</i> (see text).</p

MceX negatively regulates the expression of the microcin E492 structural gene.

<p>(A) <i>wt</i> and Δ<i>fur E</i>. <i>coli</i> cells transformed with the reporter p<i>mceBA</i>’-‘<i>lacZ</i> were grown in M9 medium supplemented with 100 μM 2,2’-Bipyridyl (low iron availability) or 10 μM FeSO₄ (high iron availability). β-galactosidase activity (expressed in Miller Units) was measured at different growth phases (Early exponential, OD₆₀₀ = 0.4–0.6; Late exponential, OD₆₀₀ = 0.9–1.1; Stationary, OD₆₀₀ = 1.9–2.1). In the construct scheme, the orange circle represents the previously determined transcription start site. (B) Wild type <i>E</i>. <i>coli</i> cells were transformed with pT5-<i>mceX</i> (allowing the IPTG-inducible expression of MceX), or with the backbone plasmid pUC57 as negative control. The activity was expressed as the percentage of β-galactosidase activity after induction respect to the negative control. Error bars represent the standard deviation between 6 independent experiments. ***<i>P</i><0.001, ****<i>P</i><0.0001, ns: not significant.</p

Fur overexpression reduces the antibacterial activity of <i>E</i>. <i>coli</i> cells carrying the gene cluster for microcin E492 production.

<p><i>E</i>. <i>coli</i> cells carrying pMccE492 (allowing the production of active MccE492) were transformed with p15A-<i>fur</i> (driving overexpression of Fur carrying a His-tag) or pACYC184 (control). (A) Two representative clones of p15A-<i>fur</i> containing cells were grown in M9 medium alone or supplemented with IPTG (1 mM), and total protein extracts were prepared after 3 and 24 h of growth. SDS-PAGE analysis of the extracts followed by Coomassie Blue staining revealed a ~17-kDa protein band largely enriched in cells grown in presence of IPTG (black arrow). Immunoblot using an anti-His antibody confirmed the induction of Fur expression in presence of IPTG, although a basal expression was detected after 24 h of growth in absence of inducer. (B) Antibacterial activity of <i>E</i>. <i>coli</i> cells producing MccE492, transformed with p15A-<i>fur</i> or a control plasmid (pACYC184). The antibacterial activity was measured as a function of the growth inhibition halo’s area over a sensitive strain layer, in M9 medium supplemented with IPTG, and then normalized to the value obtained in the control condition (plasmid backbone). Error bars indicate standard deviation from 40 measurements performed for each condition. ***<i>P</i><0.001.</p

The expression of <i>mceX</i> and the microcin E492 maturation genes <i>mceJI</i> is regulated by Fur and iron availability.

<p><i>wt</i> and Δ<i>fur E</i>. <i>coli</i> cells transformed with the reporters p<i>mceX</i>’-‘<i>lacZ</i> (A) or p<i>mceJ17</i>’-‘<i>lacZ</i> (B), were grown in M9 medium supplemented with 100 0μM 2,2’-Bipyridyl (low iron availability) or 10 μM FeSO₄ (high iron availability). β-galactosidase activity (expressed in Miller Units) was measured at different phases of growth (Early exponential, OD₆₀₀ = 0.4–0.6; Late exponential, OD₆₀₀ = 0.9–1.1; Stationary, OD₆₀₀ = 1.9–2.1). In the construct schemes, the orange circle represents the experimentally determined transcription start site and the red square represents the functional Fur box. Error bars represent the standard deviation between 6 independent experiments. ***<i>P</i><0.001, ****<i>P</i><0.0001, ns: not significant.</p