Rapid Sampling of Escherichia coli After Changing Oxygen Conditions Reveals Transcriptional Dynamics.

Escherichia coli is able to shift between anaerobic and aerobic metabolism by adapting its gene expression, e.g., of metabolic genes, to the new environment. The dynamics of gene expression that result from environmental shifts are limited, amongst others, by the time needed for regulation and transcription elongation. In this study, we examined gene expression dynamics after an anaerobic-to-aerobic shift on a short time scale (0.5, 1, 2, 5, and 10 min) by RNA sequencing with emphasis on delay times and transcriptional elongation rates (TER). Transient expression patterns and timing of differential expression, characterized by delay and elongation, were identified as key features of the dataset. Gene ontology enrichment analysis revealed early upregulation of respiratory and iron-related gene sets. We inferred specific TERs of 89 operons with a mean TER of 42.0 nt/s and mean delay time of 22.4 s. TERs correlate with sequence features, such as codon bias, whereas delay times correlate with the involvement of regulators. The presented data illustrate that at very short times after a shift in oxygenation, extensional changes of the transcriptome, such as temporary responses, can be observed. Besides regulation, TERs contribute to the dynamics of gene expression.

Transition of an Anaerobic Escherichia coli Culture to Aerobiosis: Balancing mRNA and Protein Levels in a Demand-Directed Dynamic Flux Balance Analysis.

The facultative anaerobic bacterium Escherichia coli is frequently forced to adapt to changing environmental conditions. One important determinant for metabolism is the availability of oxygen allowing a more efficient metabolism. Especially in large scale bioreactors, the distribution of oxygen is inhomogeneous and individual cells encounter frequent changes. This might contribute to observed yield losses during process upscaling. Short-term gene expression data exist of an anaerobic E. coli batch culture shifting to aerobic conditions. The data reveal temporary upregulation of genes that are less efficient in terms of energy conservation than the genes predicted by conventional flux balance analyses. In this study, we provide evidence for a positive correlation between metabolic fluxes and gene expression. We then hypothesize that the more efficient enzymes are limited by their low expression, restricting flux through their reactions. We define a demand that triggers expression of the demanded enzymes that we explicitly include in our model. With these features we propose a method, demand-directed dynamic flux balance analysis, dddFBA, bringing together elements of several previously published methods. The introduction of additional flux constraints proportional to gene expression provoke a temporary demand for less efficient enzymes, which is in agreement with the transient upregulation of these genes observed in the data. In the proposed approach, the applied objective function of growth rate maximization together with the introduced constraints triggers expression of metabolically less efficient genes. This finding is one possible explanation for the yield losses observed in large scale bacterial cultivations where steady oxygen supply cannot be warranted.

Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry.

Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk1(11A)), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis.