Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance

Abstract

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.

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

“…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).…”

Osmoregulation in Escherichia coli: complementation analysis and gene-protein relationships in the proU locus

“…Relevant restriction endonuclease cleavage sites are marked: B, BglII; E, EcoRI; H, HindIll; N, NsiI; R, EcoRV; and S, Sal. The BglII site in proU has been lost in the process of construction of pHYD58 (24) and is therefore represented in parentheses on the line corresponding to this plasmid. A kilobase scale is included; the DNA between the EcoRI and BglII sites in pHYD56 is 3.5 kb long.…”

“…The ProU transporter is involved in the active uptake of glycine betaine and of L-proline from the medium in cells subjected to water stress and in the consequent ability of these compounds, in submillimolar concentrations, to promote growth in media of otherwise inhibitory osmolarity (8,14,20,24,37). The transcription of proU is induced several hundred-fold upon growth in high-osmolarity media, and the activity of the transporter is also increased under these conditions (8,15,20,27,33).…”

“…In the context of this study, the ProU-phenotype has been defined as one that manifests, in a putPA proP strain background, as (i) resistance to 0.3 mM 3,4-DL-dehydroproline (DHP) in minimal A medium supplemented with 0.2 M NaCl (DHP-NaClr) and (ii) an inability of 1 mM glycine betaine or 1 mM L-proline to confer osmoprotection in minimal A medium supplemented with 0.65 M NaCl (20,24). The ProU+ designation refers to strains exhibiting the converse of the two phenotypes above.…”

“…lysogens; lysates of the two lac fusion phages employed for mutagenesis, Mu dII301(lac Ap) encoding Ampr and X plac Mu55 encoding Kanr, were each prepared and used as described (7,11,33 (24). Relevant restriction endonuclease cleavage sites are marked: B, BglII; E, EcoRI; H, HindIll; N, NsiI; R, EcoRV; and S, Sal.…”