An In Vivo Metabolic Approach for Deciphering the Product Specificity of Glycerate Kinase Proves that Both E. coli’s Glycerate Kinases Generate 2-Phosphoglycerate

Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions

Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement

Product specificity of glycerate kinases from <i>E. coli</i> and A. thaliana.

<p>(A) A <i>ΔgpmA ΔgpmM ΔgarK ΔglxK</i> strain was able to grow on a minimal medium supplemented with glycerol and glycerate when one of <i>E. coli’s</i> glycerate kinases were expressed, indicating that these enzymes generate 2-phosphoglycerate. (B) Growth on pyruvate and glycerate was possible only when <i>A. thaliana’s</i> glycerate kinase was expressed, indicating that this enzyme produces 3-phosphoglycerate. Cells were cultivated in 96-multiwell plates and OD measurements were taken automatically every 90 minutes. For each enzyme, we show the growth of one clone with standard errors (at each time point) that are based on three parallel cultivations. Other clones showed a similar qualitative dependence on the carbon sources with somewhat different growth yield and dynamics.</p

Evolthon: A community endeavor to evolve lab evolution.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation