Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

Abstract

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,...

Growth Rate Toxicity Phenotypes and Homeostatic Supercoil Control Differentiate Escherichia coli from Salmonella enterica Serovar Typhimurium

“…As E. coli enters stationary phase, the of pBR322 falls from Ϫ0.069 at the peak growth rate to below Ϫ0.055 as cells enter early stationary phase. This has been seen before in E. coli (43,73). But in Salmonella, there is no loss of when cells enter or exit stationary phase.…”

“…But in Salmonella, there is no loss of when cells enter or exit stationary phase. What controls the changes in supercoil levels in E. coli is not known, but the supercoiling recovery does not require new protein synthesis (73). One possibility is the GyrI gyrase inhibitory protein, which is under SOS regulatory control (60).…”

Plasmid DNA Supercoiling and Survival in Long-Term Cultures of Escherichia coli : Role of NaCl

“…In lane 5, the culture received neither NaCl nor ofloxacin. starvation was also shown to require RpoS (26). NaCl addition was not specifically required for long-term survival; exposure to short-chain n-alcohols also prevents viability loss in an rpoSdependent fashion (27).…”

“…Relaxation of pBR322 was observed at entry into stationary phase in the presence or absence of NaCl, but this relaxation was followed by an increase of supercoiling in NaClfree Luria-Bertani (LB) medium (8). Many authors have described a DNA plasmid relaxation as cells enter into stationary phase (1,9,26). Recently, stationary-phase cells were shown to display different strategies to ensure survival under conditions of nutritional stress and to regain growth quickly once nutrients become available (26); the recovery of DNA supercoiling and gyrase activity are essential to reinitiate growth.…”

“…Many authors have described a DNA plasmid relaxation as cells enter into stationary phase (1,9,26). Recently, stationary-phase cells were shown to display different strategies to ensure survival under conditions of nutritional stress and to regain growth quickly once nutrients become available (26); the recovery of DNA supercoiling and gyrase activity are essential to reinitiate growth. Gauthier et al (13) studied the cultivability of E. coli in seawater and found that long-term protection was afforded to cells by growth in medium whose osmotic pressure was increased by NaCl.…”