The proU locus encodes an osmotically inducible glycine betaine transport system that is important in the adaptation to osmotic stress. We present evidence that DNA supercoiling plays a key role in the osmotic induction of proU transcription. An increase in extracellular osmolarity increases in vivo DNA supercoiling, and the expression of proU is highly sensitive to these changes. Furthermore, topA mutations can mimic an increase in osmolarity, facilitating proU expression even in media of low osmolarity in which it is not normally expressed. Selection for trans-acting mutations that affect proU expression has yielded only mutations that alter DNA supercoiling, either in topA or a new genetic locus, osmZ, which strongly influences in vivo supercoiling. Mutations in osmZ are highly pleiotropic, affecting expression of a variety of chromosomal genes including ompF, ompC, fimA, and the bgl operon, as well as increasing the frequency of site-specific DNA inversions that mediate fimbrial phase variation.