A synthetic growth switch based on controlled expression of RNA polymerase

International audienceThe ability to control growth is essential for fundamental studies of bacterial physiology and biotechnological applications. We have engineered an Escherichia coli strain in which the transcription of a key component of the gene expression machinery, RNA polymerase, is under the control of an inducible promoter. By changing the inducer concentration in the medium, we can adjust the RNA polymerase concentration and thereby switch bacterial growth between zero and the maximal growth rate supported by the medium. We show that our synthetic growth switch functions in a medium-independent and reversible way, and we provide evidence that the switching phenotype arises from the ultrasensitive response of the growth rate to the concentration of RNA polymerase. We present an application of the growth switch in which both the wild-type E. coli strain and our modified strain are endowed with the capacity to produce glycerol when growing on glucose. Cells in which growth has been switched off continue to be metabolically active and harness the energy gain to produce glycerol at a twofold higher yield than in cells with natural control of RNA polymerase expression. Remarkably, without any further optimization, the improved yield is close to the theoretical maximum computed from a flux balance model of E. coli metabolism. The proposed synthetic growth switch is a promising tool for gaining a better understanding of bacterial physiology and for applications in synthetic biology and biotechnology

The temperature of the Icelandic mantle from olivine-spinel aluminum exchange thermometry

New crystallization temperatures for four eruptions from the Northern Volcanic Zone of Iceland are determined using olivine-spinel aluminum exchange thermometry. Differences in the olivine crystallization temperatures between these eruptions are consistent with variable extents of cooling during fractional crystallization. However, the crystallization temperatures for Iceland are systematically offset to higher temperatures than equivalent olivine-spinel aluminum exchange crystallization temperatures published for MORB, an effect that cannot be explained by fractional crystallization. The highest observed crystallization temperature in Iceland is 1399 ± 20°C. In order to convert crystallization temperatures to mantle potential temperature, we developed a model of multilithology mantle melting that tracks the thermal evolution of the mantle during isentropic decompression melting. With this model, we explore the controls on the temperature at which primary melts begin to crystallize, as a function of source composition and the depth from which the magmas are derived. Large differences (200°C) in crystallization temperature can be generated by variations in mantle lithology, a magma's inferred depth of origin, and its thermal history. Combining this model with independent constraints on the magma volume flux and the effect of lithological heterogeneity on melt production, restricted regions of potential temperature-lithology space can be identified as consistent with the observed crystallization temperatures. Mantle potential temperature is constrained to be 1480−30+37 °C for Iceland and 1318−32+44 °C for MORB.O.S. was supported by a Title A Fellowship from Trinity College Cambridge and a Geology Option Postdoctoral Fellowship at Caltech

A Coarse-Grained Biophysical Model of E. coli and Its Application to Perturbation of the rRNA Operon Copy Number

We propose a biophysical model of Escherichia coli that predicts growth rate and an effective cellular composition from an effective, coarse-grained representation of its genome. We assume that E. coli is in a state of balanced exponential steadystate growth, growing in a temporally and spatially constant environment, rich in resources. We apply this model to a series of past measurements, where the growth rate and rRNA-to-protein ratio have been measured for seven E. coli strains with an rRNA operon copy number ranging from one to seven (the wild-type copy number). These experiments show that growth rate markedly decreases for strains with fewer than six copies. Using the model, we were able to reproduce these measurements. We show that the model that best fits these data suggests that the volume fraction of macromolecules inside E. coli is not fixed when the rRNA operon copy number is varied. Moreover, the model predicts that increasing the copy number beyond seven results in a cytoplasm densely packed with ribosomes and proteins. Assuming that under such overcrowded conditions prolonged diffusion times tend to weaken binding affinities, the model predicts that growth rate will not increase substantially beyond the wild-type growth rate, as indicated by other experiments. Our model therefore suggests that changing the rRNA operon copy number of wild-type E. coli cells growing in a constant rich environment does not substantially increase their growth rate. Other observations regarding strains with an altered rRNA operon copy number, such as nucleoid compaction and the rRNA operon feedback response, appear to be qualitatively consistent with this model. In addition, we discuss possible design principles suggested by the model and propose further experiments to test its validity

Transcriptome and Proteome Exploration to Model Translation Efficiency and Protein Stability in Lactococcus lactis

This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms

RADIOACTIVE SULFUR TRACER STUDIES ON THE REPRODUCTION OF T4 BACTERIOPHAGE

In the experiments with P32, we selected for structures with the properties of the protein membrane of the phage, and then looked for phosphorus associated with them. Different results might have been obtained if the protein itself had been labeled. Such experiments have now been carried out, again using the phage T4, but labeling with S35. The results indicate that noninfective, sedimentable particles which adsorb and precipitate with the specificity of the T4 phages are present in both premature and ordinary lysates. From the negative results obtained with P32 we conclude that these particles are phosphorus-free