Metabolic responses of Escherichia coli upon glucose pulses captured by a capacitive field-effect sensor.

Living cells are complex biological systems transforming metabolites taken up from the surrounding medium. Monitoring the responses of such cells to certain substrate concentrations is a challenging task and offers possibilities to gain insight into the vitality of a community influenced by the growth environment. Cell-based sensors represent a promising platform for monitoring the metabolic activity and thus, the ‘‘welfare’’ of relevant organisms. In the present study, metabolic responses of the model bacterium Escherichia coli in suspension, layered onto a capacitive field-effect structure, were examined to pulses of glucose in the concentration range between 0.05 and 2 mM. It was found that acidification of the surrounding medium takes place immediately after glucose addition and follows Michaelis– Menten kinetic behavior as a function of the glucose concentration. In future, the presented setup can, therefore, be used to study substrate specificities on the enzymatic level and may as well be used to perform investigations of more complex metabolic responses. Conclusions and perspectives highlighting this system are discussed

Metabolic responses of Escherichia coli upon glucose pulses captured by a capacitive field-effect sensor

Living cells are complex biological systems transforming metabolites taken up from the surrounding medium. Monitoring the responses of such cells to certain substrate concentrations is a challenging task and offers possibilities to gain insight into the vitality of a community influenced by the growth environment. Cell-based sensors represent a promising platform for monitoring the metabolic activity and thus, the "welfare" of relevant organisms. In the present study, metabolic responses of the model bacterium Escherichia coli in suspension, layered onto a capacitive field-effect structure, were examined to pulses of glucose in the concentration range between 0.05 and 2 mM. It was found that acidification of the surrounding medium takes place immediately after glucose addition and follows Michaelis-Menten kinetic behavior as a function of the glucose concentration. In future, the presented setup can, therefore, be used to study substrate specificities on the enzymatic level and may as well be used to perform investigations of more complex metabolic responses. Conclusions and perspectives highlighting this system are discussed. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.status: publishe

Selection of Optimization Trials for Maximized SH Production in Recombinant Strains.

<p>Specific activities were determined in extracts of cells from three independent cultivation trials. Error bars indicated represent standard deviations. Parameters were modified as follows: <i>Temperature</i> (<b>1</b>∶18°C; <b>2</b>∶25°C; <b>3</b>∶30°C; <b>4</b>∶37°C), <i>Time</i> (<b>1</b>∶24 h; <b>2</b>∶30 h; <b>3</b>∶36 h; <b>4</b>∶42 h), <i>Medium</i> (<b>1</b>: LB; <b>2</b>: TB; <b>3</b>: HEM; <b>4</b>: M9), <i>Ferric ammonium citrate</i> (<b>1</b>∶0 µM; <b>2</b>∶50 µM; <b>3</b>∶100 µM; <b>4</b>∶500 µM), <i>C-sources</i> (given as % (wt/vol) glucose/% (vol/vol) glycerol/% (wt/vol) lactose; <b>1</b>∶0.05/1/0.2; <b>2</b>∶0.1/2/0.4; <b>3</b>∶0.1/2/0.8; <b>4</b>∶0.2/2/0.8), <i>NiCl₂</i> (<b>1</b>∶0 µM; <b>2</b>∶1 µM; <b>3</b>∶25 µM; <b>4</b>∶100 µM).</p

Current model of SH maturation in <i>Cupriavidus necator</i>.

<p>Steps: <b>1.</b> HypC binds to HoxH, preventing improper folding prior metal-center assembly <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Drapal1" target="_blank">[70]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Magalon1" target="_blank">[71]</a>; <b>2.</b> Delivery of the iron center comprising the diatomic ligands: The cyanide ligands are derived from carbamoyl phosphate (CMP) in a transcarbamoylation/dehydration reaction catalyzed by HypF/HypE, thereby transferring the carbamoyl moiety to a C-terminal cysteine of HypE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Blokesch1" target="_blank">[72]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Paschos1" target="_blank">[74]</a>. Modified HypE forms a complex with the preassembled HypCD complex, which is likely to “store” the iron complex until the ligand coordination is completed and subsequently delivers it to apo-HoxH. This step probably involves a redox reaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Brstel1" target="_blank">[75]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Soboh2" target="_blank">[76]</a>; <b>3.</b> Nickel insertion is mediated by the concerted action of HypA and HypB in a GTP dependent reaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Magalon2" target="_blank">[77]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Maier2" target="_blank">[79]</a>; <b>4.</b> HypC detaches from HoxH; <b>5.</b> HoxW cleaves a 24 amino acid peptide off the C-terminus of the HoxH apoprotein. HoxW activity requires pre-incorporated nickel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Theodoratou1" target="_blank">[80]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Thiemermann1" target="_blank">[81]</a>; <b>6.</b> HoxH folds and thereby buries the bimetallic core inside the protein (at the hydrophobic contact surface to HoxY). The SH subunits assemble; Prior to this step, FMN cofactors and the iron-sulfur clusters are delivered by the cellular machineries. Unresolved reactions, which include the action of HypX and the origin of the carbonyl ligand are indicated (*).</p

Purification table for endogenous SH purified from <i>Cupriavidus necator</i> cells.

<p>About 20 g of wet packed cells were used for purification.</p>a<p>H₂:NAD⁺physiological activity, measured under anaerobic conditions. 1 Unit is defined as the H₂-mediated reduction of 1 µmol NAD⁺per minute.</p

UV/Vis Spectroscopy of Recombinant SH.

<p>Main: Spectrum of purified, oxidized SH_var2 (1 mg·mL⁻¹); Inset: Difference spectrum between oxidized and dithionite (500 µM) reduced enzyme.</p

List of <i>Cupriavidus necator</i> genes cloned in this project.

a<p>The <i>hyd4</i> putative specific C-terminal endopeptidase encoded in PHG070 was not included in the regular pM2 constructs and its derivatives, since HoxW was in different studies proven to be the HoxH specific protease essential for catalytic activity.</p><p>CMP = Carbamoyl phosphate.</p

Gene selection for heterologous SH expression in <i>Escherichia coli</i>.

<p><b>a</b>) Distribution of hydrogenase related genes and putative SH related genes on the pHG1 megaplasmid of <i>Cupriavidus necator</i> (<i>Cn</i>). pHG1 comprises three distinct hydrogenase clusters (locus MBH cluster: 100–22390; locus <i>hyd4</i> cluster: 59962–74032; locus SH cluster: 79712–89227) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812-Schwartz1" target="_blank">[52]</a>. The MBH cluster contains the MBH and RH structural genes along with numerous accessory genes for MBH, RH and SH maturation. A partially duplicated set of maturation related genes is present in vicinity to the SH structural genes. The putative structural and auxiliary genes localized in the <i>hyd4</i> cluster have not been characterized to date. The <i>hyp</i> genes present in this cluster have been tested as an alternative set for SH maturation in this study. Thicker arrows indicate structural genes. <b>b</b>) Main expression constructs designed in this study for recombinant SH production in <i>E. coli</i>. Plasmids used for maturation and SH production trials were derivatives of the three depicted constructs. The pM1 and pM2 derivatives differ in the origin of the <i>hypABCDEF</i> genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone-0068812-t001" target="_blank">Table 1</a>). Two independent genes <i>hypX</i> and <i>hoxN1</i>, as well as the HoxH specific endopeptidase gene <i>hoxW</i>, were included in both pM1 and pM2 derivatives. All genes were placed under control of individual T7-promoters and -terminators.</p