Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region ofproV also exhibited partial derepression ofproU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.The growth rate of Escherichia coli and Salmonella typhimurium in high-osmolarity media is promoted by the addition of small concentrations of L-proline or glycine betaine to the culture medium. The osmoprotective effect of both of these compounds is presumed to be consequent upon their intracellular accumulation under conditions of water stress and is in part dependent on the presence of a functional ProU transporter in these cells, encoded by genes of the proU locus (for a review, see reference 6).Complementation studies using a number of proU mutants, in combination with nucleotide sequence analysis of the locus, have shown that proU is an operon composed of three structural genes, proV, proW, and proX (7, 13). The product of proX is a periplasmic protein which has been purified and shown to be a glycine betaine-binding protein in vitro; furthermore, the deduced amino acid sequences of the products of proV and proW each show similarities to components of other well-characterized transport systems such as those for histidine, maltose, or arabinose, thus permitting the inference t...

The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU.

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the S -and 70 -bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of S selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.The proU operon in Escherichia coli and Salmonella typhimurium encodes a binding-protein-dependent transporter that mediates the osmoprotective effects of exogenous glycine betaine and L-proline when these organisms are grown in media of elevated osmolarity. proU transcription is markedly induced (more than 100-fold) in high-osmolarity media, and the mechanism by which this is brought about has been the subject of intensive, but as yet inconclusive, genetic and biochemical studies (for reviews, see references 6, 16, and 29).With regard to the cis elements mediating proU osmoresponsivity, there is a consensus on the existence of (i) a promoter whose transcription start site is approximately 60 nucleotides upstream of the initiation codon of the first structural gene (proV) (10,15,41,46,55) and (ii) a negative regulatory element (NRE) situated in a region overlapping the proximal (5Ј) end of proV whose deletion leads to a 25-fold derepression of proU expression at low osmolarity (8,13,30,40,42). This promoter is recognized in vitro by the 70 -RNA polymerase holoenzyme (E 70 ) (10, 55). Our group has also identified, by in vivo studies, another promoter located 250 nucleotides upstream of proV in E. coli (8,15) and has shown recently that this promoter is both RpoS ( S ) dependent and stationary-phase inducible (31). We have designated the two (proV-proximal and proV-distal) promoters P2 and P1, respectively. The role of the upstream P1 promoter and S -RNA polymerase (E S ) in proU regulation is still uncertain, however, for the following reasons: (i) proU expression in vivo is not affected by deletion of P1 or by mutations in rpoS (28, 31), whereas a mutation in rpoD (encoding 70 ) results in nearly complete abolition of proU expression (58); (ii) proU expression in vivo is not significantly induced in stationaryphase cultures (31); (iii) Overdier et al. (41) have failed to identify an equivalent promoter during subcloning experiments with S. typhimurium proU; and (iv) in vi...

Unlike the 70 -controlled P2 promoter for the osmotically regulated proU operon of Escherichia coli and Salmonella enterica serovar Typhimurium, the s -controlled P1 promoter situated further upstream appears not to contribute to expression of the proU structural genes under ordinary growth conditions. For S. enterica proU P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and trans-acting mutations which activate in vivo expression from proU P1. The cryptic S. enterica proU P1 promoter was activated, individually and additively, in a rho mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10°C. The E. coli proU P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream proU sequence, and in these cases activation of in vivo expression was achieved either by a rho mutation during growth at 10°C or by an hns null mutation (affecting the nucleoid protein H-NS) at 30°C. The rho mutation had no effect at either 10 or 30°C on in vivo expression from two other s -controlled promoters tested, those for osmY and csiD. In cells lacking the RNA-binding regulator protein Hfq, induction of E. coli proU P1 at 10°C and by hns mutation at 30°C was still observed, although the hfq mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from proU P1 is modulated both by nucleoid structure and by Rhomediated transcription attenuation and that this promoter may be physiologically important for proU operon expression during low-temperature growth.The ProU transporter in Escherichia coli and Salmonella enterica serovar Typhimurium is a binding-protein-dependent transport system that mediates the cytoplasmic accumulation of compatible solutes such as glycine betaine, L-proline, and related compounds during growth of cells in media of elevated osmolarity (9, 10). The subunit polypeptides of the transporter are encoded by three genes, proV, proW, and proX, which together constitute (in that order) the proU operon (16).Transcription of proU in both E. coli and S. enterica is activated several-hundredfold in cultures grown in high-osmolarity media, but the mechanism of osmotic induction of the operon is not fully understood (reviewed in references 10, 19, and 29). Two cis regulatory elements that have been identified (see Fig. 1) include a 70 -driven promoter whose transcription start site is situated 60 bases upstream of proV (16, 24, 28, 54) and a negative regulatory element (NRE) approximately 500 bp long, which is situated downstream of the promoter (overlapping the proV coding region) and whose deletion results in partial derepression of proU at low osmolarity (11,24,37,38). Mutations in hns, the gene encoding an abundant nucleoid protein, H-NS, also result in partial derepression of proU expression (for a review of H-NS...

RpoS ( S ) inEscherichia coli is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial 70 family of proteins. Conserved domain 1.1 at the N-terminal end of 70 has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a S variant with a deletion of its N-terminal 50 amino acids ( S ⌬1-50), when expressed in vivo either from a chromosomal rpoS::IS10 allele (in rho mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the proU P1 promoter; at three other S -dependent promoters that were tested (osmY, katE, and csiD), the truncated protein exhibited a three-to sevenfold reduced range of activities. Catabolite repression at the csiD promoter (which requires both S and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing S ⌬1-50. The intracellular content of S ⌬1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for S , even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type rpoS. Our results indicate that, unlike that in 70 , the N-terminal domain in S may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of S -specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of S protein degradation.

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the S -and 70 -bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by S RNA polymerase in vitro and that a 22-bp deletion from ؉63 to ؉84 (relative to the start site of transcription) confers S -dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to ؉60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to ؉85 is able to do so only from the latter. Our results suggest that the S -controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site.Adaptation of microorganisms to growth in media of elevated osmolarity (osmoregulation) is associated with the cytoplasmic accumulation of nontoxic low-molecular-weight organic compounds that are collectively designated compatible solutes. In the enterobacteria, the list of identified compatible solutes includes trehalose, glutamate, L-proline, and glycine betaine (reviewed in reference 4). The proU operon in Escherichia coli and Salmonella typhimurium encodes a binding-protein-dependent transporter that mediates the accumulation of glycine betaine and L-proline in response to hyperosmotic stress. Transcription of proU in both organisms is induced several-hundred-fold under these conditions (reviewed in references 4, 9, and 16).In E. coli, two promoters, P1 and P2, have been identified for proU, with start sites of transcription approximately 250 and 60 bp, respectively, upstream of the first structural gene, proV. A negative regulatory element, situated downstream of P2 and overlapping the proximal end of proV, is also required in cis to repress transcription of proU in low-osmolarity media (reviewed in references 9 and 16). Data from both genetic and in vitro transcription experiments indicate that the P1 and P2 promoters are transcribed by RNA polymerase holoenzymes bearing, respectively, the S (that is, stationary-phase sigma factor) and the 70 polypeptides (17,23,28,32). The finding that proU has a S -dependent promoter that is induced in stationary-phase cultures (17) is in keeping with the overlap that has been observed between osmotic and stationary-phasespecific gene regulation at several loci in E. coli (14).The mechanism of proU regulation in S. typhimurium is very similar to that in E. coli, with one exception. Overdier and Csonka (21) were unable to identify promoter activity equivalent to that of the S -regulated E. coli P1 during subcloning experiments with S. typhimurium proU, despite the fact that sequence resid...