Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proUlacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-4acZ fusion gene at low copy number showed that the plasmidencoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also induced some proU expression, but other salts were ineffective, thereby ruling out ionic strength as the stimulatory signal. High concentrations of sucrose, trehalose, or glycine betaine did not induce proU expression in vitro either, eliminating osmolarity per se as the stimulus. Reconstitution in a cell-free system rules out osmoregulatory mechanisms that depend on turgor, transmembrane signaling, or trans-acting regulators synthesized after osmotic upshock.

proU expression has been proposed to form part of a general stress response that is regulated by increased negative DNA supercoiling brought about by environmental signals such as osmotic or anaerobic stress (N. Ni Bhriain, C. J. Dorman, and C. F. Higgins, Mol. Microbiol. 3:933-944, 1989). However, we find that although proU-containing plasmids derived from cells grown in media of elevated osmolarity were more supercoiled than plasmids from cells grown in standard media, they did not activate proU expression in vitro. The gyrA96 mutation and anaerobic conditions are known to affect DNA supercoiling but did not alter proU expression. Finally, the gyrase inhibitors coumermycin and novobiocin did not reduce in vitro proU expression. Therefore, this evidence rules out regulation by changes in DNA superhelicity for proU in Escherichia coli.

No abstract

No abstract

The presence of RNase activity has been detected in the two saxicolous lichen species, Lasallia hispanica (Frey) Sancho & Crespo and Cornicularia normoerica (Gunn.) DR. Activity was localized in the soluble fraction and had an acid optimum pH in both species. When proteins from the soluble fraction of the two lichens were separated by isoelectric focusing, multiple electromorphs with RNase activity were detected. L. hispanica RNase was separated into seven bands, characterized by pis 7, 6.28, 4.58, 4.45, 4.25, 3.95, and 3.47. In C. normoerica four bands were detected, with pis of 6.28, 3.98, 3.57, and 3.39. The molecular mass of the main RNase of L. hispanica estimated by SDS-PAGE was 31.86 kDa, which corresponds to the 33 kDa estimated for the undenatured RNase by gel chromatography. Proteins from C. normoerica were resolved by SDS-PAGE in three bands, with estimated molecular mass of 36.07 kDa, 31.86 kDa and 17.13 kDa. In order to improve the detection of RNase activity, gels were incubated after the run (electrophoresis or isoelectric focusing) in a RNA solution, instead of including the substrate in the gel. In both species, RNase activity increased during hydration and decreased during desiccation. This pattern of activity resembles that of other enzyme activities in lichens, which decrease in response to water deficits, and is different from the response of other poikilohydrous organisms such as bryophytes. These results are discussed in relation to the mechanisms that lichens have to withstand dehydration.