Molecular Microbiology (1989) 3{7), 933-942 An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression N. Ni Bhriain,* C. J. Dorman and C. F. Higgins Molecular Genetics Laboratory. Department ot Bioctiemistry, Dundee University, Dundee DD1 4HN, UK. Summary The regulation of several genes in response to osmotic and anaerobic stress has been examined. We have demonstrated a clear overlap between these two regulatory signals. Thus, the osmotically induced proU and ompC genes require anaerobic growth for opti- mum induction while the anaerobically induced tppB gene is also regulated by osmolarity. Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions. Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expres- sion of the osmoticalty regulated proU gene. The oxrC mutation is shown to affect the level of negative supercoiling of ptasmid DNA and its effects on gene expression can be explained as secondary con- sequences of altered DNA topology. We suggest that there is a class of 'stress-regulated' genes that are regulated by a common mechanism in response to different environmental signals. Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses. Introduction There is now considerable evidence for global regulatory networks in which a given environmental signal causes the coordinate induction or repression of a number of diverse and unlinked genes (Gottesman, 1984). For example, when cells are shifted from aerobic to anaerobic growth conditions the rate of synthesis of over fifty proteins is altered (Smith and Neidhardt. 1983a,b; Spector ef al.. 1986). Similarly, the expression of many genes is affected by changes in the osmoiarity or pH of the growth medium (Higgins ef a/., 1987; Guitierrez ef a/., 1987; Aliabadi ef a/., 1988). Unlike more specific regulatory stimuli, such as the presence or absence of a defined nutrient, these more Received 11 January, 1989; revised 7 March, 1989. 'For correspondence. 'diffuse' environmental changes have often proved less tractable when it comes to identifying regulatory proteins which mediate the cell response. For example, although the FNR protein was identified many years ago as a positive regulator of a number of anerobically induced genes (Newman and Cole, 1978; Lambden and Guest, 1976), it is now clear that many anaerobic genes are FNR-independent. Additional regulatory loci, each affect- ing a different subset of anaerobically induced genes. have been identified (Jamleson and Higgins, 1984; 1986; Aliabadi ef al.. 1988). Similarly, selections designed to identify genes responsible for osmotic control of gene expression have failed to Identify a global osmotic regula- tory protein (Higgins et al.. 1988). Although the OmpR and EnvZ proteins are important for the osmotic regulation of porin expression (Hall and Siihavy, 1981a,b), the ex- pression of most osmotically regulated genes is OmpR- and EnvZ-independent (Cairney ef ai. 1985; May et al., 1986; Gultierrez etal., 1987). DNA supercoiling is known to play an important role in determining the efficiency with which many promoters are transcribed. Two enzymes, DNA gyrase and DNA topo- isomerase I, are primarily responsible for determining the level of DNA supercoiling. DNA gyrase adds negative supercoils in an energy-dependent reaction while topo- isomerase I removes supercoils and relaxes DNA. Pertur- bation of the activities of these enzymes, by mutation or by the use of inhibitors, affects transcription from a variety of promoters (Sanzey, 1979; Driica, 1984; 1987; Richardson etal., 1984; Menzel and Gellert, 1987; Jovanovich and Lebowitz, 1987) and many promoters are sensitive to DNA supercoiling in v/(ro (Wood and Lebowitz, 1984; Borowiec and Gralla, 1987; Dixon etai, 1988). Recently it has been demonstrated that the supercoiling of cellular DNA is not maintained at a constant level but varies in response to growth conditions. Thus, environmental stresses such as osmolarity or anaerobicity can influence the supercoiling of cellular DNA (Higgins et al., 1988; Dorman et al., 1988). Other factors such as growth phase and carbon limitation also affect DNA topology (Dorman et al.. 1988; Baike and Gralla, 1987). Furthermore, these changes in DNA super- coiling appear to be responsible for regulating the ex- pression of a number of specific genes in response to environmental stimuli (Higgins etal., 1988; Dorman etal.. 1988). The finding that different environmental stresses