DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase.

DNA of prokaryotes is in a nonequilibrium structural state, characterized as 'active' DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell 34, 105-113]. We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem. 266, 865-877]. Of the two regulatory mechanisms via which homeostasis is conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).     

ECA: control in ecosystems

Although metabolic control analysis (MCA) cannot be applied directly to microbial ecological systems because of mass conservation and stoichiometric constraints, we demonstrate here that Hierarchical Control Analysis (HCA) can be applied to such systems. We illustrate the approach for a particular ecosystem example of the biological synthesis of acetic acid from glucose, and uncover some surprising aspects to the control of this miniature ecosystem.     

Monitoring bacterial growth using tunable resistive pulse sensing with a pore-based technique

A novel bacterial growth monitoring method using a tunable resistive pulse sensor (TRPS) system is introduced in this study for accurate and sensitive measurement of cell size and cell concentration simultaneously. Two model bacterial strains, Bacillus subtilis str.168 (BSU168) and Escherichia coli str.DH5α (DH5α), were chosen for benchmarking the growth-monitoring performance of the system. Results showed that the technique of TRPS is sensitive and accurate relative to widely used methods, with a lower detection limit of cell concentration measurement of 5?×?10⁵ cells/ml; at the same time, the mean coefficient of variation from TRPS was within 2 %. The growth of BSU168 and DH5α in liquid cultures was studied by TRPS, optical density (OD), and colony plating. Compared to OD measurement, TRPS-measured concentration correlates better with colony plating (R?=?0.85 vs. R?=?0.72), which is often regarded as the gold standard of cell concentration determination. General agreement was also observed by comparing TRPS-derived cell volume measurements and those determined from microscopy. We have demonstrated that TRPS is a reliable method for bacterial growth monitoring, where the study of both cell volume and cell concentration are needed to provide further details about the physical aspects of cell dynamics in real time.     

Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells

Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.     

Thymidylate synthetase inhibitors and fragile site expression in lymphocytes.

The ability of three thymidylate synthetase inhibitors, fluorodeoxyuridine, fluorodeoxycytidine, and trifluorothymidine, to induce the expression of eight different folate-sensitive fragile sites has been investigated in 22 patients and compared with the efficacy of simple folate deprivation for inducing fragile site expression. Fluorodeoxyuridine and fluorodeoxycytidine were equal in their ability to elicit fragile site expression but fluorodeoxycytidine proved less cytotoxic under comparable culture conditions. Both fluorodeoxyuridine and fluorodeoxycytidine were found to be more efficient than trifluorothymidine at comparable concentrations but less efficient than simple folate deprivation in eliciting fragile site expression in lymphocytes. Since the three inhibitors induced expression of eight different folate-sensitive fragile sites, it is likely that all folate-sensitive fragile sites have a common underlying mechanism of expression. The practical application of thymidylate synthetase inhibitors in the routine detection of heritable fragile sites is discussed.