The make-up and function of a particular cell reflects the spatio-temporal organization of the expression of its genetic information as encoded in the DNA. However, gene expression is not controlled in a dictatorial manner, but determined by a quasi-democratic interaction with processes at all levels in the cellular hierarchy [Westerhoff et al., 1990a]. In particular, the living cell is regulated in response to many of the various external stimuli it receives via the plasma membrane. The hierarchical control aHierarchical Control Analysis (HCA) method aims at elucidating how this regulation proceeds along various routes. It involves the subtle manipulation of molecular processes in the intact cell, and quantitative measurements of the consequences both at the level of the process itself and at the physiological level of the intact cell. In a nutshell, membrane linked free energy transduction consists of the inter-conversion of three forms of Gibbs energy. These are (i) the redox potential of the NADH/NAD couple (and any of a set of other redox couples, which are not necessarily at equilibrium with the NAD(H) couple) relative to oxygen (or other electron acceptors) (DGox), (ii) the electrochemical potential difference for protons across the plasma membrane, (DmH+), and (iii) the Gibbs energy of hydrolysis of ATP (DGp). Plasma membrane proton pumps, components of the so-called electron transfer chain and the H+-ATPase, interconvert these free energies. The Gibbs energy residing in DGp and DmH+ is applied to energetically uphill biosynthetic reactions and transport. DGox, DGp, and even DmH+ can also be supplemented more directly by ca?abolism of growth substrates. Until recently, regulation of energy transduction in microbial physiology was studied almost exclusively in terms of direct interactions between these processes. The role of gene expression herein was conceived of as in terms of a dictatorial hierarchy, neglecting the possibility of a feedback from the level of the three energy potentials to transcription of the genes. The group of Westerhoff has pointed at an important candidate for regulation of transcription by DGp: the high energy structure of the prokaryotic DNA. Prokaryotic DNA is supercoiled by the action of DNA gyrase under concomitant hydrolysis of ATP. Westerhoff and colleagues have demonstrated that in vitro the extent of DNA supercoiling achieved by DNA gyrase is strongly sensitive to the magnitude of DGp [Westerhoff et al., 1988]. When the energy state in vivo is compromised by various methods, both DGp and DNA supercoiling decrease [Westerhoff et al., 1990; Westerhoff and Van Workum, 1990; Hsieh et al., 1988; Drlica et al., 19911991ab; Jensen et al., 1995; Van Workum et al., 1995]. Transcription of most E. coli operons is sensitive to DNA supercoiling [Gellert, 1981]. Importantly, these sensitivities extend to the promoters of DNA gyrase and topoisomerase I (which is the enzyme that removes supercoils from DNA) [review: Drlica et al., 1990]. It will be the aim of this research program to examine how strong the regulatory loop from membrane-linked free energies through DNA gyrase, DNA supercoiling and transcription to cell function (and back to free-energy transduction) is relative to the already known direct regulatory interactions at the level of energy meabolism. The relative importance of the regulatory routes through triose phosphates on RNA polymerize will also be examined.