Intracellular F-actin reorganization by is dependent on the Lys of SteC

<p><b>Copyright information:</b></p><p>Taken from "SteC is a kinase required for SPI-2-dependent F-actin remodelling"</p><p></p><p>Cellular Microbiology 2008;10(1):20-30.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253691.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> A. Representative confocal images of Swiss 3T3 fibroblasts infected for 8 h with an mutant containing plasmid-borne , or . Cells were immunolabelled to detect (green in merged image). F-actin was visualized by phalloidin-RRX staining (red in merged image). Bars represent 5 μm. B. Quantification of F-actin reorganization by strains. Results are the means ± SE of three independent experiments, in which a total of 300 infected cells were examined for each strain. Results for cells infected with wild-type or mutant strains are for comparison and are from . C. Representative confocal images of infected cells showing translocation of SteC-2HA and SteCK256H-2HA expressed from a low-copy plasmid. Cells were fixed and immunolabelled to detect (green in merged image) and HA (red in merged image). Bars represent 5 μm. D. Representative confocal micrograph of an infected cell showing translocated SteC-2HA localizing to F-actin. Cells were fixed and immunolabelled to detect (blue in merged image) and HA (green in merged image. F-actin was visualized by phalloidin-RRX staining (red in merged image). Boxed insets are magnifications of indicated region showing localization of SteC-2HA with F-actin. Bars represent 5 μm

P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> MG1655 transformed with empty vector, native, constitutive or hybrid <i>phaCAB</i> constructs were cultured in 1 liter LB media, supplemented with 3% glucose (w/v) for 24 hours or 48 hours. P(3HB) was purified from these cultures and measured as <b>(A)</b> P(3HB) production (g/L) and <b>(B)</b> P(3HB) content (weight [wt.] % of cell dry weight [CDW]). Data represent the mean +/- the standard deviation of three independent experiments. Student t-test, *P<0.05, **P <0.01, ***P <0.001 and ****P <0.0001.</p

Flow cytometry analysis of P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i> from waste-media cultures.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either empty vector [EV], native [N], constitutive [C] or hybrid [H] <i>phaCAB</i> constructs were cultured in 5 ml of waste-media for 36 h at 37°C. P(3HB) content was assessed via flow cytometry analysis of Nile Red staining. (<b>A</b>) Representative forward scatter (FSC) and side scatter (SSC) contour plots. (<b>B</b>) Representative histogram (FL-5). (<b>C</b>) Normalized fluorescence of Nile Red stained <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, from three independent experiments. Error bars, +/- the standard deviation. Student t-test, *P<0.05 and ***P <0.001.</p

<i>phaCAB</i> pathway and constructs.

<p>(<b>A</b>) Schematic of poly-3-hydroxybutyrate (P(3HB)) production via the <i>phaCAB</i> operon pathway. (<b>B</b>) The constructs used in this study. Abbreviations: Pwt (wildtype promoter; green arrow), J23104 (Anderson constitutive promoter, BBa_J23104; red arrow), B0034 (ribosomal binding site, BBa_B0034; red half-circle), <i>phaC</i> (PHA synthase), <i>phaA</i> (3-ketothiolase) and <i>phaB</i> (acetoacetyl-CoA reductase). Green half-circles denote native ribosomal binding sites. Construct symbols are based on the Synthetic Biology Open Language Visual (SBOLv) v1.0.0 guidelines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117202#pone.0117202.ref021" target="_blank">21</a>].</p

Simulated P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p>In order to simulate P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, a P(3HB) synthesis model was constructed using the Simbiology toolbox of Matlab. Using this model the flux of several metabolites and species were simulated in order to identify aspects of the system that could be selectively tuned to increase the production of P(3HB). From these analyses, several novel <i>phaCAB</i> operons were designed. These data show the simulated P(3HB) production across several different <i>phaCAB</i> operon designs, where <i>phaCAB</i> expression is under the control of the indicated Anderson constitutive promoters.</p