Proc. Nati. Acad. Sci. USA Vol. 86, pp. 1153-1157, February 1989 Biochemistry In vitro reconstitution of osmoregulated expression of proU of Escherichia coli (glycine betaine/lac fusion/osmotic stress) R. M. RAMIREZ*, W. S. PRINCE*, E. BREMERt, AND M. VILLAREJO*t *Department of Biochemistry and Biophysics, University of California, Davis, CA 95616; and tFakultat fur Biologie, Universitat Konstanz, D-7750 Konstanz 1, Federal Republic of Germany Communicated by Eugene P. Kennedy, November 4, 1988 (received for review June 6, 1988) ABSTRACT Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmopro- tectant glycine betaine in Escherichia coli. Previously, a proU- lacZ 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 plasmid- encoded 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 pro- gram protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also in- duced 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, trans- membrane signaling, or trans-acting regulators synthesized after osmotic upshock. In Escherichia coli, as in many other bacteria (1), the primary response to increased medium osmolarity is the influx and accumulation of K+. To a first approximation, intracellular potassium concentration increases in proportion to external osmolarity (2). K+ transport in E. coli is mediated by two genetically distinct transport systems, Trk and Kdp (3). The high-affinity Kdp system is induced by a reduction in cellular turgor pressure measured by a transmembrane osmosensor, KdpD and -E (4, 5). Stressed cells also accumulate large amounts of glutamate, which may act as a counterion to K+ for maintaining electroneutrality (6, 7). The net result is that K+ glutamate is the predominant ionic species within the osmotically stressed cell. Elevated K+ concentration appar- ently affects E. coli adversely, since cells shocked with salt concentrations greater than 0.2 M NaCl exhibit decreased growth rates (8). Osmoprotective compounds, such as cho- line (9), proline (10), or glycine betaine (11) restore the cell's ability to grow in osmotically stressful environments. Glycine betaine uptake in E. coli and Salmonella typhimurium is mediated by two transport systems, ProP and ProU (12-14). proP encodes a constitutive, low-affinity transport system whose expression is stimulated severalfold during osmotic upshock (12, 14). proU encodes a high-affinity system that is strongly induced at the transcriptional level by elevated osmolarity (8, 12-14). Both systems are also regulated at the level of transport activity (12-15). In E. coli, one gene product of the ProU system has been purified and charac- terized. It is a periplasmic polypeptide that specifically binds glycine betaine (16). Most studies of proU regulation have utilized genetic fusions with lacZ (14, 17). 83-Galactosidase activity from FproU-lacZ fusions is induced 60- to 100-fold by osmotic upshock (8, 13). The mechanism of proU osmoregulation is unknown, although intracellular K+ concentration has been proposed as the signal stimulating gene expression (17). Addition of exogenous glycine betaine represses proU expression (8, 14). Glycine betaine is accumulated to high intracellular concentrations under osmotic stress, replacing K+ as the major osmolyte (11, 18). The mechanism of glycine betaine repression of proU is also undefined. Osmotic stress stimulates the expression of a number of genes (19, 20), one of which specifies the outer membrane porin OmpC (21). Expression of another porin, OmpF, is affected in a reciprocal manner (21); both porins respond to a two-protein regulatory system encoded by the unlinked ompB operon composed of envZ and ompR (22). EnvZ is an inner membrane protein sensor whereas OmpR, a cytoplas- mic protein, is a positive regulator of ompC and ompF transcription (23). Expression of proU, kdp, and ompC are all stimulated by high osmolarity, yet they fundamentally differ with respect to their regulatory mechanisms. Whereas kdp expression is controlled by changes in turgor pressure (5) and is repressed by K+, elevated proU expression correlates with high intra- cellular levels of K+ (17). Unlike ompC, proU expression is not dependent on envZ or ompR (13, 14). Furthermore, extensive mutant searches have not revealed the involvement of any unlinked specific regulatory loci controlling proU expression (24, 25). Thus, the precise nature of the signal and mechanism by which proU expression is induced by high osmolarity remains unknown. In this study, we report the in vitro reconstitution of osmoregulated proU expression in a cell-free S-30 system. These results faithfully reproduce effects seen in vivo and further define the signals and mechanism controlling proU expression during osmotic stress. MATERIALS AND METHODS Strains, Plasmids, and Growth Conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Standard synthetic medium contained M9 salts (28), 0.4% glycerol, and thiamine (1 ug/ml). Typically, a stationary-phase culture, grown in LB medium (28) containing an appropriate antibi- otic, would be used to inoculate an overnight M9/glycerol culture, grown aerobically at 37°C with agitation. Cells from the latter were then used to inoculate fresh M9/glycerol medium. After resuming growth and upon reaching midlog- arithmic phase (":-45 Klett units; red filter), cells were osmotically upshocked either by the addition of 5 M NaCl to tTo whom reprint requests should be addressed. 1153 The publication costs of this article were defrayed in part by page charge payment. 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