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

Abstract

The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.The ability to maintain cellular integrity during long-term starvation is essential for the survival of bacteria. Such starvation occurs upon entry into stationary phase. Once thought to be characterized by metabolic inactivity, stationary phase is now recognized as a period during which a series of metabolic changes take place over time (17,19,24).The enteric bacterium Escherichia coli has to cope with continuous change in its environment. One of the variable parameters is the osmolarity of the surrounding medium. In E. coli, the level of supercoiling is maintained by the opposing actions of DNA gyrase and topoisomerase I, and it is increased when cells are cultured in medium of high salt concentration (16,22) or in anaerobiosis (15). This laboratory previously reported that the level of negative supercoiling of pBR322 changed during growth according to the absence or presence of NaCl in the medium. 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. 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. Survival in seawater was correlated to the topological state of the DNA. Apart from a study of cultivability of E. coli in river water (12), little attention has been paid to long-term survival in the absence of NaCl. We previously reported poor longterm survival of E. coli grown in NaCl-free LB medium compared to the good viability of cells grown in rich media supplemented with either 170 or 400 m...

Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS–ArlR two-component system of Staphylococcus aureus

“…8a). In E. coli, plasmid DNA relaxes as cells enter the stationary phase (Conter, 2003). Similarly, we observed DNA relaxation when cells entered stationary phase, even though the expression of topoisomerase genes is not modified at this growth stage (data not shown), suggesting that other factors such as cellular energy (ATP) are also involved in the regulation of DNA supercoiling in S. aureus.…”

“…The agr (Morfeldt et al, 1988;Peng et al, 1988) and sarA (Cheung et al, 1992) loci are the best characterized of these loci. Several other regulatory loci have also been described: sar homologues , 2003McNamara et al, 2000;Schmidt et al, 2001;Tegmark et al, 2000), sae (Giraudo et al, 1994), arl (Fournier et al, 2001) and srr (Yarwood et al, 2001). In S. aureus, virulence determinant production is modulated in response to growth conditions (Chan & Foster, 1998a, b;Lindsay & Foster, 1999;Novick, 2000), reflecting the ability of S. aureus to adapt and to survive in many different environmental niches.…”

E. coli Fis Protein Insulates the cbpA Gene from Uncontrolled Transcription

“…Numerous studies have shown that entry to stationary phase results in a marked decrease in DNA supercoiling [2], [30], [31]. Thus, we investigated the effect of CbpA, and as a control Dps, on DNA topology in vivo using a reporter plasmid.…”

Overcoming the production limitations of Photorhabdus temperata ssp. temperata strain K122 bioinsecticides in low-cost medium

“…It was obviously clear that in P. temperata strain K122, NaCl at The values presented are the average of three replicates of three separate experiments 5 g/l allowed partial overcome of the VBNC state, since culturability reached 31.1 and 7.5% of the total cells, counted microscopically, instead of 11.1 and 4.35% obtained in the optimized medium and the complex medium, respectively. Culturability improvement through NaCl addition was demonstrated to be correlated with the topological state of the plasmid DNA in E. coli cells [22]. Indeed, E. coli cells grown in the presence of NaCl exhibited a relatively mild relaxation of plasmid DNA, contrarily to those cultured in the absence of NaCl that showed abrupt relaxation of plasmid DNA accompanied by the inability to form colonies in the solid medium.…”