Precise Switching of Flagellar Gene Expression in Escherichia Coli by the FlgM–FliA Regulatory Network

A remarkable feature of flagellar synthesis in Escherichia coli is that gene expression is sequential and coupled to the assembly process. The interaction of two key proteins, the flagellar sigma factor FliA and its anti-sigma factor FlgM serves as a major checkpoint in the assembly process that temporally separates middle and late gene expression. While the sequential nature within each gene class has been studied using large-scale transcriptional data, much less is known about the timing controlled by the checkpoint mechanism. In this article, we analyze timing, sensitivity and robustness of the FlgM–FliA core regulatory mechanism based on quantitative molecule data and a detailed stochastic as well as reduced deterministic reaction kinetics model. We find that the pool of free anti-sigma factor FlgM, accumulated during middle gene expression, acts as a molecular timer that determines the delay between successful completion of the hook basal body subunit and the start of expression of flagellar filament proteins. Furthermore, we find that the number of free FliA molecules needs to be tightly controlled for a precise switch from middle to late gene expression. A sensitivity analysis based on the reduced reaction kinetics model reveals that the checkpoint mechanism is very sensitive to changes in levels of competing sigma factors, allowing the bacterium to rapidly adapt to a changing environment. In addition, we find that the reduced model also shows a high sensitivity to the effective synthesis rates of FliA and FlgM. However, this high sensitivity does not generally carry over to the original parameters of transcriptional and translational processes in the detailed model. As a consequence, care has to be taken whenever interpreting results from the robustness analysis of reaction kinetic models comprising lumped or effective parameters

CAEv–A program for computer aided evaluation

The evaluation of new reagents and instruments in clinical chemistry leads to complex studies with large volumes of data, which are difficult to handle. This paper presents the design and development of a program that supports an evaluator in the definition of a study, the generation of data structures, communication with the instrument (analyser), online and offline data capture and in the processing of the results. The program is called CAEv, and it runs on a standard PC under MS-DOS. Version 1 of the program was tested in a multicentre instrument evaluation. The concept and the necessary hardware and software are discussed. In addition, requirements for instrument/host communication are given. The application of the laboratory part of CAEv is described from the user's point of view. The design of the program allows users a high degree of flexibility in defining their own standards with regard to study protocol, and/or experiments, without loss of performance. CAEv's main advantages are a pre-programmed study protocol, easy handling of large volumes of data, an immediate validation of the experimental results and the statistical evaluation of the data