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M Villarejo

Publications and source records attributed to M Villarejo.

At least 19 recordsLinked to original sources
PubMed1996

Sequences in the -35 region of Escherichia coli rpoS-dependent genes promote transcription by E sigma S.

sigma S is an alternate sigma factor which functions with RNA polymerase to activate transcription of genes that are involved in a number of stress responses, including stationary-phase survival and osmoprotection. The similarity of the sigma S protein to sigma D (Escherichia coli's major sigma factor) in the regions thought to recognize and bind promoter sequences suggests that sigma S- and sigma D-associated RNA polymerases recognize promoter DNA in a similar manner. However, no promoter recognition sequence for sigma S holoenzyme (E sigma S) has been identified. An apparent conservation of cytosine nucleotides was noted in the -35 region of several sigma S-dependent promoters. Site-directed mutagenesis and reporter gene fusions were used to investigate the importance of the -35 cytosine nucleotides for sigma S-dependent transcription. Substitution of cytosine nucleotides for thymidine at the -35 site of the sigma D-dependent proU promoter effectively abolished transcription by E sigma D but allowed E sigma S to direct transcription from the mutant promoter. Inclusion of the sigma D consensus -10 hexamer strengthened transcription by E sigma S, demonstrating that both E sigma D and E sigma S can recognize the same -10 sequences. Conversely, replacement of -35 site cytosine nucleotides with thymidine in the sigma S-dependent osmY promoter reduced transcription by E sigma S and increased transcription by E sigma D. Our data suggest that DNA sequences in the -35 region function as part of a discriminator mechanism to shift transcription between E sigma D and E sigma S.

Amino Acid Transport Systems↗
PubMed1995

Promoter selectivity control of Escherichia coli RNA polymerase by ionic strength: differential recognition of osmoregulated promoters by E sigma D and E sigma S holoenzymes.

Transcription in vitro of two osmoregulated promoters, for the Escherichia coli osmB and osmY genes, was analysed using two species of RNA polymerase holoenzyme reconstituted from purified core enzyme and either sigma D (sigma 70, the major sigma in exponentially growing cells) or sigma S (sigma 38, the principal sigma at stationary growth phase). Under conditions of low ionic strength, the osmB and osmY promoters were transcribed by both E sigma D and E sigma S. Addition of up to 400 mM potassium glutamate (K glutamate), mimicking the intracellular ionic conditions under hyperosmotic stress, specifically enhanced transcription at these promoters by E sigma S but inhibited that by E sigma D. At similar high concentrations of potassium chloride (KCl), however, initiation at both these promoters was virtually undetectable. These data suggest that the RNA polymerase, E sigma S, itself can sense osmotic stress by responding to changes in intracellular K glutamate concentrations and altering its promoter selectivity in order to recognize certain osmoregulated promoters.

DNA-Directed RNA Polymerases↗
PubMed1995

Two different Escherichia coli proP promoters respond to osmotic and growth phase signals.

proP of Escherichia coli encodes an active transport system for proline and glycine betaine which is activated by both hyperosmolarity and amino acid-limited growth. proP DNA sequences far upstream from the translational start site are strongly homologous to the promoter of proU, an operon that specifies another osmoregulated glycine betaine transport system. Mutation and deletion analysis of proP and primer extension experiments established that this promoter, P1, was responsible for proP's strong expression in minimal medium and its response to osmotic signals. When cells were grown in complex medium, expression from a proP-lacZ fusion was induced three- to fourfold as growth slowed and cells entered stationary phase. Stationary-phase induction was dependent on rpoS, which encodes a stationary-phase sigma factor. Deletion of 158 bp of the untranslated leader sequence between P1 and the proP structural gene abolished rpoS-dependent stationary-phase regulation. Transcription initiation detected by primer extension within this region was absent in an rpoS mutant. proP is therefore a member of the growing class of sigma S-dependent genes which respond to both stationary-phase and hyperosmolarity signals.

Bacterial Proteins↗
PubMed1994

Molecular characterization of the promoter of osmY, an rpoS-dependent gene.

The osmY gene, which encodes a periplasmic protein with an apparent M(r) of 22,000, is induced by both osmotic and growth phase signals. We demonstrate here that osmY expression is regulated at the level of transcription and that transcription initiates 242 nucleotides upstream of the osmY open reading frame. Relative to the transcriptional start site, 5' deletions up to -36 did not inhibit osmY expression. 3' deletions that extended into the untranslated leader region affected the overall level of osmY::lacZ expression but did not affect inducibility. 5' and 3' deletions that extended past the transcriptional start region essentially abolished osmY expression, suggesting that there is a single promoter region. A putative promoter was identified, and its -10 region, TATATT, closely resembles the sigma 70 consensus -10 sequence, TATAAT. However, we show that osmY is not absolutely dependent on a functional sigma 70 for its expression. Since osmY expression does require rpoS (R. Hengge-Aronis, R. Lange, N. Henneberg, and D. Fischer, J. Bacteriol. 175:259-265, 1993), which encodes a stationary-phase sigma factor, sigma S (K. Tanaka, Y. Takayanagi, N. Fujita, A. Ishihama, and H. Takahashi, Proc. Natl. Acad. Sci. USA 90:3511-3515, 1993), E sigma S may be the form of RNA polymerase responsible for transcription of osmY.

Bacterial Proteins↗
PubMed1994

The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation.

The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock. Regulation occurs at the transcriptional level. KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock. Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity. Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals. A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible. However, transcription from this fragment was resistant to inhibition by salt shock. A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter. The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal.

Amino Acid Transport Systems↗
PubMed1994

Genes encoding osmoregulatory proline/glycine betaine transporters and the proline catabolic system are present and expressed in diverse clinical Escherichia coli isolates.

Sixty-three clinical isolates identified as Escherichia coli, 30 from the human urinary tract and 33 derived from other human origins, were screened for proline/glycine betaine transporters similar to those that support proline catabolism and proline- or glycine betaine-based osmoregulation in E. coli K-12. Both molecular (DNA- and protein-based) analyses and physiological tests were performed. All tests were calibrated with E. coli K-12 derivatives from which genetic loci putP (encoding a proline transporter required for proline catabolism), proP, and (or) proU (loci encoding osmoregulatory proline/glycine betaine transporters) had been deleted. All clinical isolates showed both enhanced sensitivity to the toxic proline analogue azetidine-2-carboxylate on media of high osmolality and growth stimulation by glycine betaine in an artificial urine preparation of high osmolality. DNA sequences similar to the putP, proP, and proU loci of E. coli K-12 were detected by DNA amplification and (or) hybridization and protein specifically reactive with antibodies raised against the ProX protein of E. coli K-12 (a ProU constituent) was detected by western blotting in over 95% of the isolates. Two anomalous isolates were reclassified as non-E. coli on the basis of the API 20E series of tests. A protein immunochemically cross-reactive with the ProP protein of E. coli K-12 was also expressed by the clinical isolates. Since all three transporters were ubiquitous, no particular correlation between clinical origin and PutP, ProP, or ProU activity was observed. These data suggest that the transporters encoded in loci putP, proP, and proU perform housekeeping functions essential for the survival of E. coli cells in diverse habitats.

Amino Acid Transport Systems↗
PubMed1992

Growth of Escherichia coli in human urine: role of salt tolerance and accumulation of glycine betaine.

Glycine betaine is a powerful osmoprotectant molecule present in the inner medulla of the kidney and excreted into urine. It may be responsible for the ability of Escherichia coli to grow in hypertonic urine. Also, strains of E. coli that cause urinary tract infections may be more salt-tolerant than strains from other sites. To explore these questions, 301 isolates from blood, urine, or stool and 12 representative enteric strains were examined. Tolerance varied from 0.1 to 0.7 M NaCl (median, 0.5) in minimal medium. There were no significant differences in salt tolerance by site of isolation. A salt-sensitive enteric strain that responded poorly to glycine betaine and mutant strains lacking the ability to synthesize or transport glycine betaine did not grow well in hypertonic urine. Accumulation of glycine betaine appears to be a mechanism by which E. coli can adapt to external osmotic forces and grow in hypertonic urine.

Betaine↗
PubMed1992

osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli.

A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.

Amino Acid Sequence↗
PubMed1991

Osmotic signal transduction to proU is independent of DNA supercoiling in Escherichia coli.

proU expression has been proposed to form part of a general stress response that is regulated by increased negative DNA supercoiling brought about by environmental signals such as osmotic or anaerobic stress (N. Ni Bhriain, C. J. Dorman, and C. F. Higgins, Mol. Microbiol. 3:933-944, 1989). However, we find that although proU-containing plasmids derived from cells grown in media of elevated osmolarity were more supercoiled than plasmids from cells grown in standard media, they did not activate proU expression in vitro. The gyrA96 mutation and anaerobic conditions are known to affect DNA supercoiling but did not alter proU expression. Finally, the gyrase inhibitors coumermycin and novobiocin did not reduce in vitro proU expression. Therefore, this evidence rules out regulation by changes in DNA superhelicity for proU in Escherichia coli.

Anaerobiosis↗
PubMed1990

Transcription of osmB, a gene encoding an Escherichia coli lipoprotein, is regulated by dual signals. Osmotic stress and stationary phase.

The osmB gene, which encodes an outer membrane lipoprotein, can be induced by both osmotic and growth phase signals. Construction of two transcriptional fusions, an osmB-lacZ fusion in single copy on the bacterial chromosome and an osmB-cat fusion carried on a multicopy plasmid, demonstrated that induction of osmB by hyperosmolarity and during the stationary phase of growth occurred at the level of transcription. Two transcription initiation sites were identified by RNase protection of in vivo message. The downstream P2 promoter is the primary site for regulation; the basal level of expression is initiated at P2 and transcription from P2 is induced by elevated osmolarity or upon reaching stationary phase. Transcription from the P1 promoter, 150 base pairs (bp) upstream of the P2 promoter, occurred only when both osmotic and growth phase signals were present simultaneously; that is, when cells growing in high osmolarity medium have reached stationary phase. Deletion analysis narrowed the sequences necessary for P2 regulation to the 42-bp region upstream from the transcription start site. A 7-bp sequence just upstream from the -35 region was identified as a cis-acting regulatory element essential for osmotic stimulation of osmB expression. A hexanucleotide sequence within this segment could form the left arm of a region of dyad symmetry, flanking the -35 region of the promoter. Stationary phase induction at P2 does not require the 7-bp element.

Bacterial Outer Membrane Proteins↗
PubMed1989

In vitro reconstitution of osmoregulated expression of proU of Escherichia coli.

Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant 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-lacZ 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 program protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also induced 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.

Chromosomes, Bacterial↗
PubMed1987

Purification and characterization of a glycine betaine binding protein from Escherichia coli.

A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000. The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine. This periplasmic protein has now been purified to homogeneity. Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein. The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport. The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.

Amino Acid Sequence↗
PubMed1986

Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12.

Glycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity. We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region. We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 microM. ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression. The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity. We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity. Like ProU, this system is regulated at two levels: both gene expression and the activity of the transport system are osmotically stimulated. Using lambda plac Mu-generated lacZ operon and gene fusions, we find that expression of the proU region is osmotically regulated at the level of transcription. We cloned a part of the proU region together with the phi(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.

Bacteriophage lambda↗
PubMed1986

Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli.

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.

Adaptation, Physiological↗
PubMed1985

Coordinate expression of a small polypeptide with the lactose carrier of Escherichia coli.

Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16-kDa inner membrane protein in addition to the known products of the lacZ, lacY, and lacA genes. Cells carrying the lacY gene on a multicopy plasmid overproduce this 16-kDa polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, [35S]methionine labeling of minicells carrying the lacY plasmid shows that the 16-kDa protein is not synthesized from the plasmid DNA. We have purified and partially characterized the 16-kDa protein. It is an acidic membrane protein of apparent Mr = 15,800 whose amino-terminal sequence (NH2-Met-Arg-Asn-Phe-Asp-Leu-) does not match any known lac operon DNA sequence. Using antibody prepared to the purified 16-kDa protein, we have quantitatively analyzed conditions under which this protein is made and have shown that the amount of 16-kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligodeoxyribonucleotide probe complementary to the 5' end of 16-kDa protein mRNA shows that its synthesis is regulated at the level of transcription.

Amino Acid Sequence↗
PubMed1984

envZ mediates transcriptional control by local anesthetics but is not required for osmoregulation in Escherichia coli.

Expression of a particular set of exported protein genes (ompF, ompC, phoA, and malE) can be specifically altered in three ways: variation in the osmotic strength of the growth medium, mutations in the regulatory locus envZ, or treatment with sublethal concentrations of the local anesthetic procaine. To clarify relationships among these factors in the regulation of transcription, expression of the affected genes was compared in envZ+ and envZ22(Am) mutant strains grown in media of differing osmolarities with and without procaine. Loss of the envZ product resulted in complete resistance of gene expression to procaine, supporting the hypothesis that envZ mediates procaine inhibition. The specific activity of the phoA product, alkaline phosphatase, was elevated in envZ22 mutant strains, while the amounts of both the OmpC and OmpF porins were reduced. Osmotic control of phoA and ompC was retained in the absence of envZ function, but osmoregulation of ompF was lost. Therefore, the envZ product is somehow involved in the complex regulation of all four target genes, but is not solely responsible for osmoregulation.

Bacterial Proteins↗
PubMed1983

Osmoregulation of alkaline phosphatase synthesis in Escherichia coli K-12.

Alkaline phosphatase, the phoA product, is synthesized constitutively in phoR mutants. This constitutive synthesis, which is independent of phosphate control, varies with changes in the osmolarity of the growth medium; phoA expression increases with increasing osmolarity. Maximum expression of the osmoregulated genes phoA, ompC, and ompF was achieved by osmotic manipulation of minimal medium; complex media repressed their expression.

Alkaline Phosphatase↗