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At least 307 records · Page 17Linked to original sources
PubMed1983

Characterization of the operator sites of the exu regulon in Escherichia coli K-12 by operator-constitutive mutations and repressor titration.

In Escherichia coli, the exu regulon of the hexuronate system involves the three exuT, uxaCA and uxaB operons and is under the negative control of the exuR regulatory gene product. The technique developed by Casadaban, Chou and Cohen was employed to construct two plasmids containing operon fusions in which the lactose genes were fused to the uxaCA and exuT operons. These fusions were transferred into the chromosome by a reciprocal recombination event, and the resulting strains were used for isolation of mutants defective in repression. Two types of operator-constitutive mutants were obtained: one specific for the uxaCA operon expression and the other affecting the exuT gene expression. This genetic evidence confirms that these two operons which are divergently transcribed each possess their own operator site.--The derepressed expression of the two exuT-lac and uxaCA-lac operons and the uxaB gene was also examined upon introduction of plasmids bearing various operators of the exu regulon. The results of testing exuR repressor titration by multiple copies of the exu operators allowed us to show a gradation in the affinity degrees for the three exu operators: uxaBo has the strongest affinity for the exuR repressor and uxaCo the weakest, although that of exuTo seems to be just slightly greater. This gradation may play a role in the control of the exu regulon expression.

Chromosomes, Bacterial↗
PubMed1985

Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids.

We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected by cAMP. The inhibition of cya expression by cAMP was also observed in the cya-lac fused operon system, although it was necessary to increase the amount of cAMP or CRP in the cells to detect the effect. The results indicate that cAMP-CRP is a negative regulator of cya expression at the level of transcription.

Adenylyl Cyclases↗
PubMed2001

Different characteristics distinguish early versus late arising adaptive mutations in Escherichia coli FC40.

The Escherichia coli strain FC40 has frequently been employed to investigate the mechanism of adaptive mutations. The strain cannot utilize lactose due to a +1 frameshift mutation that reduces beta-galactosidase to about 1% of normal levels. Cells undergo a high rate of mutation from Lac- to Lac+ when cells are grown with lactose as the sole energy source. Almost all Lac+ colonies arising 3-6 days after plating result from a base pair deletion in runs of iterated base pairs within a 130-bp target region. In this study we characterized Lac+ colonies arising 3-10 days after plating. Temperature gradient gel electrophoresis (TGGE) was used to detect mutations in the target region as a function of the day a colony appears. TGGE results confirmed the occurrence of mutations within the target region in 36 of 37 FC40 Lac+ colonies arising on days 3-7. However, mutations in this region were not detected in 23 of 37 Lac+ colonies arising from days 8-10. Sequencing data verified the TGGE results. Half of the Lac+ mutants arising on days 8-10 with no base pair change in the target region were unstable and exhibited a Lac- phenotype after successive growth cycles in rich medium. The results suggest that amplification of the lac operon region is a common factor in late arising colonies, and that different characteristics distinguish early and late arising Lac+ colonies.

Adaptation, Biological↗
PubMed2002

Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity.

Expression systems based on the Escherichia coli arabinose operon P(BAD) promoter exhibit the all-or-nothing (autocatalytic) induction of expression that was first documented in the lac operon. Under conditions of subsaturating levels of inducer, some of the cells of the population are fully induced, whereas other cells remain uninduced. Recently, a new AraE transporter system was reported to have circumvented the problem of autocatalytic expression in the pBAD expression vectors and to provide graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations [Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A. & Keasling, J. D. (2000) J. Bacteriol. 182, 7029-7034]. However, we report that nonuniform gene expression in the AraE system was readily detectable by the use of mutant green fluorescent proteins that are rapidly degraded in E. coli. We report an approach to avoid all-or-nothing induction of the pBAD promoter; the use of a mutant LacY transporter in a strain deficient in both arabinose transport (araE araFGH) and degradation (araBAD). This mutant LacY protein performs facilitated diffusion of arabinose resulting in homogeneous expression of an unstable GFP that is maintained over extended incubation times at subsaturating levels of inducer. This approach is readily adapted to other sugar-regulated expression systems.

Arabinose↗
PubMed1984

Enhanced recombination between lambda plac5 and mini-F-lac: the tra regulon is required for recombination enhancement.

F42lac recombination with lambda plac is normally 20-fold to 50-fold higher than recombination between lambda plac and a chromosomal lac gene. Transductional crosses with lambda plac and a recombinant plasmid containing the lac operon and F replication functions show only two to four-fold higher recombination than similar crosses with a chromosomal lac gene. Insertion of a BamHI fragment containing the entire tra regulon of F into the mini-F-lac plasmid restores the high level of recombination seen with F42lac.

Bacteriophage lambda↗
PubMed1989

Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.

Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained approximately 2 x 10(7) active repressor molecules (and 1-2 x 10(4) copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of beta-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the beta-galactosidase gene under control of the optimal lacO promoter was constructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable beta-galactosidase. Addition of isopropyl beta-D-thiogalactoside restored expression to greater than 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.

DNA, Recombinant↗
PubMed1980

Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter.

High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter. The resolved products are RNA species of various lengths which correspond to the initial lac mRNA sequence. Quantitation shows that many oligonucleotides can be formed per preinitiation complex, including species as long as hexanucleotide. Synthesis occurs without dissociation of the enzyme, as evidenced by levels of synthesis in the presence of heparin, a selective inhibitor of free RNA polymerase. Thus, RNA polymerase cycles at this promoter in vitro producing oligonucleotides reiteratively. In general, the yield of oligonucleotides decreases when the total concentration of all four substrates is increased or when a missing nucleoside triphosphate substrate is added. Nevertheless, oligonucleotide synthesis persists under all conditions tested. Strikingly, the dinucleotide always represents 50% of the total of all oligonucleotides, even when conditions are manipulated to cause a 100-fold variation in this total. This shows that, after formation of the first phosphodiester bond at the lac UV5 promoter, dissociation of the dinucleotide is as likely as formation of the second phosphodiester bond. As discussed above, after release of a small RNA, RNA polymerase may then begin another RNA chain, which is again subject to premature release. These considerations lead to a model in which RNA polymerase cycles to produce oligonucleotides during initiation of transcription at the lac UV5 promoter in vitro. Production of a long RNA transcript is then essentially an escape from this cycling reaction. The drug rifampicin, which drastically inhibits escape to produce RNA, limits, but dose not prevent, the cycling reaction.

DNA-Directed RNA Polymerases↗
PubMed2004

Modelling DNA loops using continuum and statistical mechanics.

The classical Kirchhoff elastic-rod model applied to DNA is extended to account for sequence-dependent intrinsic twist and curvature, anisotropic bending rigidity, electrostatic force interactions, and overdamped Brownian motion in a solvent. The zero-temperature equilibrium rod model is then applied to study the structural basis of the function of the lac repressor protein in the lac operon of Escherichia coli. The structure of a DNA loop induced by the clamping of two distant DNA operator sites by lac repressor is investigated and the optimal geometries for the loop of length 76 bp are predicted. Further, the mimicked binding of catabolite gene activator protein (CAP) inside the loop provides solutions that might explain the experimentally observed synergy in DNA binding between the two proteins. Finally, a combined Monte Carlo and Brownian dynamics solver for a worm-like chain model is described and a preliminary analysis of DNA loop-formation kinetics is presented.

Base Sequence↗
PubMed1998

Unequal fidelity of leading strand and lagging strand DNA replication on the Escherichia coli chromosome.

We have investigated the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. This possibility of differential replication fidelity arises from the distinct modes of replication in the two strands, one strand (the leading strand) being synthesized continuously, the other (the lagging strand) discontinuously in the form of short Okazaki fragments. We have constructed a series of lacZ strains in which the lac operon is inserted into the bacterial chromosome in the two possible orientations with regard to the chromosomal replication origin oriC. Measurement of lac reversion frequencies for the two orientations, under conditions in which mutations reflect replication errors, revealed distinct differences in mutability between the two orientations. As gene inversion causes a switching of leading and lagging strands, these findings indicate that leading and lagging strand replication have differential fidelity. Analysis of the possible mispairs underlying each specific base pair substitution suggests that the lagging strand replication on the E. coli chromosome may be more accurate than leading strand replication.

Alleles↗
PubMed2002

Regulation and adaptive evolution of lactose operon expression in Lactobacillus delbrueckii.

Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the beta-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (beta-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus.

Adaptation, Physiological↗
PubMed1994

Quality and position of the three lac operators of E. coli define efficiency of repression.

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.

Base Sequence↗
PubMed1987

Estimation of the effect of coumermycin A1 on Salmonella typhimurium promoters by using random operon fusions.

We have estimated the extent to which relaxation of supercoiling by the DNA gyrase inhibitor coumermycin A1 affects gene expression in vivo in Salmonella typhimurium. We isolated a set of Mu d1-8 Lac+ operon fusions to random promoters and measured the effect of coumermycin A1 on the expression of 67 fusions. The differential rate of synthesis was increased for 70% of the fusions and decreased for 16%, and 13% of the fusions had less than a 25% change in expression. The coumermycin A1 response was found to correlate well (P = 0.067) with the basal level of expression such that coumermycin A1 tended to stimulate fusions with low expression and inhibit those with high expression. Since the vast majority of the fusions were sensitive to coumermycin A1 addition and, therefore, to the level of supercoiling, these results indicate that if the level of supercoiling were to vary under physiological conditions, then major readjustments in the cellular economy would occur.

Aminocoumarins↗
PubMed1985

Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter.

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.

Cations↗
PubMed1994

Nucleotide and deduced amino acid sequences of Rhizobium meliloti 102F34 lacZ gene: comparison with prokaryotic beta-galactosidases and human beta-glucuronidase.

The nucleotide (nt) sequence of a 2.57-kb Sau3A fragment carrying the Rhizobium meliloti beta-galactosidase (beta Gal)-encoding gene (RmlacZ) was determined. An open reading frame (ORF) of 2.26 kb was identified which encoded a 755-amino-acid (aa) polypeptide with a calculated molecular mass of 84,141 Da, in fair agreement with the value of 88 kDa determined by SDS-PAGE. The deduced N-terminal aa sequence was confirmed by direct sequencing of electrophoretically purified R. meliloti beta Gal. The size of the native R. meliloti beta Gal was approx. 174 kDa. Similarities were found between the aa sequence of the R. meliloti beta Gal and those from Clostridium thermosulfurogenes EM1 and Agrobacterium radiobacter, as well as human beta-glucuronidase (beta Glu). Comparisons with beta Gal from Escherichia coli, Klebsiella pneumoniae, Lactobacillus bulgaricus and Kluyveromyces lactis found only weak similarities; however, the putative active site residues appear to be conserved. The RmlacZ sequence is flanked by two partially sequenced ORFs, which show aa sequence and organisational similarities to the previously reported lac operon in A. radiobacter.

Amino Acid Sequence↗
PubMed1980

Cloning of Lac+ BamHI fragment into transposon Tn3 and transposition of the Tn3[lac] element.

By use of recombinant DNA techniques, we have inserted the lac+ operon into a transposon (Tn3). We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac+ phenotype. Our results showed that transposition of the Tn3[lac+] element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.

Cloning, Molecular↗
PubMed1987

Supercoiling facilitates lac operator-repressor-pseudooperator interactions.

The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density. However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities. Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined. These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction. The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA. Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h). These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.

DNA, Bacterial↗
PubMed1986

Analysis of the occurrence of promoter-sites in DNA.

We show that the occurrence and homology score (1) of promoter-sites in DNA depends upon the base composition of the DNA. We used simple probability theory to calculate the mean homology score expected for all promoter-sites that had a specific match in the canonical hexamers. By using the square root of this mean score as a measure of significance, we objectively classify all promoter-sites which are reported. We tested the theoretical approach in two ways. First, we used the program (PROMSEARCH) to analyze approximately 150,000 base pairs of random sequence DNA with different base compositions and we found excellent agreement with the theoretical predictions. Our second test was the analysis of a number of sequences drawn from the GENBANK DNA sequence database. We have analyzed 20 bacterial and bacteriophage sequences, which consisted of at least one operon, for promoter-sites. We found no absolute preference for promoter-sites within noncoding regions. We show the results of analyzing the phages lambda, T7 and fd, and the E. coli lac operon. The major known promoters in these sequences were all found correctly. We discuss the question of the location of a number of minor promoter-sites and show how PROMSEARCH can be used to help identify the correct location of the promoter. This approach can be applied to the search for any DNA site and should allow greater objectivity when comparing DNA sequences for meaningful subsequences.

Bacteriophage lambda↗