Characterization of mutations affecting the osmoregulated proU promoter of Escherichia coli and identification of 5' sequences required for high-level expression

Abstract

Expression of the Escherichia coli proU operon, which encodes an efficient uptake system for the osmoprotectant glycine betaine, is strongly increased in cells grown at high osmolarity. We isolated 182 independent spontaneous mutants with elevated expression of the chromosomal 4'lproV-lacZ) (Hyb2) fusion at low osmolarity. Genetic analysis demonstrated that eight of these mutant strains carried mutations closely linked to the fusion, whereas all others carried mutations that appeared to be in osmZ. All of the mutations resulted in increased but still osmoregulated expression of the fI(proV-lacZ)(Hyb2) fusion. The proU-linked mutants carried an identical point mutation (proU603) which changes the -35 sequence of the proU promoter from TTGCCT to TTGACT and thereby increases the homology of the -35 region to the consensus sequence (TTGACA) of E. coli promoters. We also selected for mutants with decreased expression of the plasmid Escherichia coli and Salmonella typhimurium can adapt to high-osmolarity growth conditions by a variety of mechanisms (for recent overviews, see references 8 and 55). One of these mechanisms is the intracellular accumulation of the osmoprotectant glycine betaine, which is either synthesized from exogenously provided choline (28) or taken up from the environment (43). Two glycine betaine porters have been identified: the low-affinity ProP system (5, 32, 34) and the high-affinity ProU system (2,6,24,32).ProU is a binding-protein-dependent transport system and is encoded by the proU operon, which consists of three structural genes, proV, proW, and proX (20, 31, 41, 52). The level of proU expression is sensitively determined by the osmolarity of the growth medium. The basal transcription of proU is very low and is strongly stimulated upon a sudden osmotic upshock. The increased steady-state level of proU expression during growth at elevated osmolarity is directly correlated with the osmolarity of the growth medium (3,6,8,13,19,21,32

Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli

“…The following lines of evidence indicate that binding of HNS to the NRE is associated with repression ofproU expression at low osmolarity. (i) proU expression is derepressed in hns mutants (6,17,23,24,31) and in cis mutants in which the NRE is deleted (6,24,29,31), whereas constructs lacking both HNS and the NRE are derepressed to the same extent as either of the single mutants (6,24,31). These experiments also demonstrated that P2R regulation is not significantly affected in hns mutants (6,24,31).…”

“…One of the Lac' clones (designated GJ1003) was shown to have a mutation unlinked to proU that caused a two-to threefold derepression of proU::lac expression. The mutation was subsequently shown to be cotransducible with the trp locus at 27 min and also to confer a Bgl+ phenotype, properties characteristic of mutations in the hns (osmZ) locus (17,23). With the aid of a linked TnlO insertion (from CAG18455) (33), the hns mutation (designated hns-201) was transduced into GJ134, and the resultant strain was designated GJ1083.…”

Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene

“…The results are somewhat complex to interpret, but the following conclusions may be drawn: (i) Plasmid pHYD275, carrying the region of proU from 376 to 552, does express the gene for P-galactosidase, and enzyme specific activity is increased sixfold at high osmolarity. Therefore, there is an osmotically regulated promoter (designated P1) in this segment, which is different from the one (designated P2) with the transcription start site at 628 characterized earlier (13,26,29).…”

“…In addition to serving as part of the proV structural gene, the region implicated above in the negative control of proU expression carries a short open reading frame on the opposite (that is, bottom) DNA strand which could conceivably encode a 70-residue peptide (beginning with a GTG codon complementary to base positions 929 to 927 on the top strand and terminating at a TAA stretch complementary to bases 719 to 717 [13]); this putative peptide has five S(T)PXX stretches in its sequence, a motif that has earlier been described to be especially prevalent in DNA-binding proteins (43). We considered the possibility that this region of DNA encodes a repressor protein involved in osmotic regulation of proU expression, because such an explanation would also account for (i) the failure of earlier attempts to identify a regulatory gene unlinked to proU (6,26) and (ii) the observation that the presence of proU on a multicopy plasmid does not lead to titration effects on regulation of the operon (14,41).…”

“…downstream of an EcoRV site in the locus, within the region whose nucleotide sequence was determined (7,13). One osmotically regulated proU promoter, with a start site of transcription 60 nucleotides upstream of the initiation codon of proV, has been identified in both E. coli and S. typhimurium (13,26,29,33,41). Primer extension analysis of proU mRNA had also indicated the existence of osmotically regulated transcripts extending as much as 250 nucleotides upstream of the proV gene (13), but no substantiating evidence for a promoter so far upstream has been reported.…”