W.C. van Heeswijk

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli

The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild‐type strain. We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII. Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD‐dependent fashion. Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a σN (σ54) RNA polymerase‐binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two‐component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII. Possible regulatory implications of this conditional redundancy of PII are discussed.

Crystallization and preliminary X-ray analysis of Escherichia coli GlnK.

The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.

Hierarchies in control

The living cell functions by virtue of an enormous number of different processes. It is one of the most difficult challenges of modern biology to elucidate how all those processes are coordinated quantitatively so as to lead to a viable system with optimal responses to various changes in the environment. The biochemical and biophysical processes of the living cell do not constitute a network with random connections. In this paper we shall discuss that cell function is organized in hierarchical substructures. We will briefly touch on the topics of (i) metabolic control and regulated gene expression, (ii) time dependent metabolism in intact yeast cells, and (iii) metabolite channelling.

Hierarchical Control of Electron-Transfer

In this chapter the role of electron-transfer in determining the behaviour of the ATP synthesising enzyme in E. coli is analysed. It is concluded that the latter enzyme lacks control because of special properties of the electron-transfer components. These properties range from absence of a strong back pressure by the protonmotive force on the rate of electron-transfer to hierarchical regulation of the expression of the genes that encode the electron-transfer proteins as a response to changes in the bioenergetic properties of the cell. The discussion uses Hierarchical Control Analysis as a paradigm. This allows one to analyse a complex system of regulatory interactions in terms of the importance of the contributing factors.

Dynamic aspects of cascade-type metabolic regulation

Cascade-type regulation, where certain enzymes in response to physiological signals modify the activity of other enzymes by covalent modification, is found in many organisms. We study the covalent regulation of glutamine synthetase which is involved in ammonia fixation in the bacterium Escherichia coli. In this paper we pose the question whether this type of regulation of glutamine synthetase has, under certain growth conditions an advantage over other types of regulation, e.g., allosteric regulation. We propose that the relatively slow dynamics of cascade-type regulation has an evolutionary advantage under conditions of fluctuating ammonia concentrations.