Evidence for transcription attenuation rendering cryptic a sigmaS-dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium

Abstract

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...

In Vivo Expression from the RpoS-Dependent P1 Promoter of the Osmotically Regulated proU Operon in Escherichia coli and Salmonella enterica Serovar Typhimurium: Activation by rho and hns Mutations and by Cold Stress

“…As explained above, all lac expression values were obtained in, and are reported for, pairs of isogenic rpoS ϩ and rpoS derivatives, whose difference has been taken to represent the in vivo activity of the RpoS-dependent proU P1 promoter under the particular test conditions; by these criteria, the plasmid vectors pMU575 and pMU2385 carrying the promoterless lacZ reporter gene displayed insignificant RpoS-dependent lac expression under any of the conditions tested in this study (data not shown). It may also be noted that the in vivo proU P1 activation studies were not amenable to analysis by mRNA primer-extension experiments, in light of our earlier findings (16,42) that even the cryptic promoter in both S. enterica and E. coli exhibits normal transcription initiation.…”

“…In an earlier study (42), we had shown that the in vivo expression from an S. enterica proU P1 promoter fragment extending from Ϫ66 to ϩ117 (S. enterica P1) of the lacZ reporter gene borne on a very low-copy-number plasmid (pHYD373) is prevented because of transcription attenuation occurring some distance downstream of the site of transcription initiation. Attenuation was relieved, and lacZ expression was consequently observed, in strains carrying a mutant plasmid derivative (pHYD374) that had suffered a 22-bp deletion between nucleotides ϩ63 and ϩ84 relative to the transcription start site (S. enterica P1⌬att).…”

“…1]) have been described earlier (11,42). Plasmids pHYD275 (E. coli P1.a) and pHYD373 (S. enterica P1) carry the isolated P1 promoters from E. coli (Ϫ60 to ϩ117) and S. enterica (Ϫ66 to ϩ117), respectively.…”

“…Attenuation was not observed in a defined in vitro transcription system, leading to the suggestions that the phenomenon is factor dependent and therefore that, under particular culture conditions (hitherto unidentified), attenuation is relieved and P1 may be able to transcribe the proU structural genes (42).…”

“…The two promoters are 190 bp apart and are oriented to transcribe in the same direction, that is, toward the proU structural genes. Data from in vivo and in vitro studies have shown that P1 transcription is moderately osmoresponsive and absolutely dependent on the stationaryphase sigma factor RpoS or s (11,16,31,42,43). However, the physiological function of P1 is unclear for the reasons that (i) cis constructs that carry P2 with the NRE but have P1 deleted continue to exhibit normal osmotic regulation of reporter gene expression (28), (ii) cis-or trans-acting mutations that affect P2 activity abolish all expression from constructs that carry both P1 and P2 (34,60,61), and (iii) proU osmotic regulation is unaffected in rpoS mutants that lack RpoS (31).…”