Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.DNA supercoiling is essential for DNA metabolism. Supercoiling is introduced into DNA molecules by enzymes called DNA topoisomerases, which break, pass DNA strands through the break, and rejoin DNA (6,24,30). In Escherichia coli the level of supercoiling depends mainly on the activities of DNA topoisomerase I (TopI) and TopII (gyrase) and, to a lesser extent, on TopIV (6,30,32). TopI introduces DNA singlestrand breaks and relaxes DNA molecules. Gyrase, an ATPdependent enzyme composed of two GyrA and two GyrB subunits, makes double-strand breaks and introduces supercoils into DNA. TopIV is an ATP-dependent enzyme that makes double-strand breaks and contributes to DNA relaxation (24,30,32). The level of DNA supercoiling is regulated by a complex homeostatic control (19,28,32). Transcription of topA, which encodes TopI, increases when the level is high, whereas transcription of the gyr genes increases when the level is low (19,28). It is well established that changes in the level of DNA supercoiling influence the activity of many promoters (9,30) and that environmental variations alter this level (2,4,8,22,23). The role of DNA topoisomerases during the cellular response to these variations is not completely understood.E. coli stationary-phase cells in minimal or rich media, or after extreme nutrient downshifts, display a relaxation of DNA (2,8,26,28). In general, stationary-phase cells exhibit a number of morphological and physiological changes in order to survive starvation. These changes include resistance to several harmful conditions, condensation of the nucleoid, increased protein degradation, and a general decrease in transcription and translation (13). In E. coli many of these characteristics depend on the product of rpoS, S , a global regulator responsible for the induction of more than 50 genes (11,12). It has been shown that S -dependent promoter recognition is more efficient on relaxed DNA templates, suggesting that the DNA relaxation that occurs in stationary-phase cells plays a role in the transcription of S -dependent genes (14). As a first step to a better understanding of the regulation of DNA supercoiling during nutritional stress, the level of supercoiling and the expression of the gyr genes were determined along the growth curve and in stationary-phase cells after dilution into fresh medium. The role of S on the level of DNA supercoiling was also explored.Variations in the level of DNA supercoiling in exponentially growing cells, cells in stationary phase, and cells starved for several hours were determined by using plasmid pMS01,...

The presence of CC(A/T)GG sequences with methylated internal cytosine (Dcm methylation) was determined in DNA from different genera of eubacteria. This methylation was studied by using restriction enzymes EcoRII and BstNI, which cleave unmethylated or methylated CC(A/T)GG sequences. Dcm methylation was only detected in genera of the family Enterobacteriaceae closely related to Escherichia: Shigella, Citrobacter, Salmonella, and Klebsiella.

The excimer-forming fluorophore dipyrenylpropane has been used to measure the relative fluidity of total membranes isolated from Escherichia coli grown at 30 or 45 degrees C, or exposed to a heat-shock from 30 to 45 degrees C for various periods of time. Parallel experiments were performed using [35S]methionine pulse-labeling of cells, to study the induction of heat-shock proteins (HSPs) at different times after the sudden change in E. coli growth-temperature from 30 to 45 degrees C. Results suggest that upon an abrupt temperature upshift from 30 to 45 degrees C, membrane fluidity adjustment to the steady-state level at the high temperature, takes place during the E. coli heat-shock response.

The possible changes in the fatty acid profile of Escherichia coli during heat‐shock have been investigated. Bacteria growing in steady‐state at 30°C were subjected to an abrupt temperature upshift to 45°C and held at the high temperature for various periods of time in order to elicit the heat‐shock response. Fatty acid compositions of lipids extracted from samples taken at different times after the temperature upshift, as well as from cultures in steady‐state at 30 and 45°C, were determined by gas‐chromatography. It has been found that the total unsaturates to total saturates ratio decreases gradually during heat‐shock and that 30 min after the temperature jump, the reduction is equivalent to 57% of the difference between ratios corresponding to steady‐state cultures at 30 and 45°C. Consistent with this remodeling of lipid acyl chains, there is a decrease in the excimerization rate of the fluidity probe dipyrenylpropane incorporated into sonicated E. coli lipid extracts. Such modifications occur within the time‐span of the heat‐shock response, as judged from our previous measurements of the kinetics of change in heat‐shock proteins induction ratio. Together, these results indicate that the control of membrane fluidity during the heat‐shock response can be accounted for, at least in part, by an important change in the fatty acid composition of Escherichia coli lipids.

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