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At least 73 records · Page 4Linked to original sources
PubMed2005

[Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

Bacteriophage lambda↗
PubMed1991

Characterization of lip expression in Salmonella typhimurium: analysis of lip::lac operon fusions.

Strains of Salmonella typhimurium which have an auxotrophic requirement for lipoic acid were isolated by mutagenesis with the transposable element Mu dJ. The chromosomal location of these insertion mutations was determined to be at 14 map units by bacteriophage P22-mediated cotransduction. The lip gene is transcribed in the clockwise direction relative to the S. typhimurium genetic map. Strains with lip::lac operon fusions were used to characterize the transcriptional activity of the lip promoter. Transcription of the lip gene is not regulated by catabolite repression or lipoic acid concentration. The data indicate that the lip gene product is expressed constitutively at a low level.

Amino Acids↗
PubMed1987

Activation of the lac operon of Escherichia coli by a mutant FNR protein.

The FNR protein of E. coli is a transcriptional activator required for the expression of genes involved in anaerobic respiratory pathways. Site-directed mutagenesis was used to alter three amino acids in the recognition helix of the putative DNA-binding domain of FNR, with the aim of changing its specificity to that of the cyclic AMP receptor protein (CRP). In the presence of the mutant protein (FNR-215) expression of the lac operon was activated during anaerobiosis and unaffected by glucose. FNR-215 did not have a uniform effect on the expression of other cAMP-CRP-dependent genes, but the results demonstrate the fundamental similarity between FNR- and CRP-mediated transcriptional activation.

Amino Acid Sequence↗
PubMed1983

The protein burden of lac operon products.

A new approach to measuring the slowing of growth due to the manufacture of proteins not needed by a bacterium is presented. An entire single colony of Escherichia coli was used to start a chemostat culture that was then given a selective pressure by the addition of phenylgalactoside (phi-gal). This enriched the population for constitutive mutants that produced beta-galactosidase without induction and could split phi-gal, consume the galactose, and grow faster. When the phi-gal was removed, the constitutives grew slower than the parental strain and were gradually lost. This procedure allows competition experiments to be carried out with minimum effects due to genetic drift. Experiments with both strains having wild-type and mutant permease genes were conducted. With the former the selective disadvantage was initially much greater than expected from the simplest hypothesis that extra unused proteins would slow growth in proportion to their fraction of the total protein synthesis. This phase was followed by a second phase where the selective disadvantage was smaller than predicted by this simple hypothesis. With a very slowly reverting permease negative strain the selective disadvantage, and therefore the protein burden, was found to be much smaller and not statistically different from zero. Thus, while one would expect under carbon and energy limitation in the chemostat the protein burden to be larger than under unlimited conditions, it is so small that even the refined technique used here could not measure it accurately. It is certainly less than the fraction of 'waste' protein synthesis; but it could be between zero and the fraction of the cells' energy and carbon budget spent on manufacture of the proteins of the lac operon.

Bacterial Proteins↗
PubMed1987

Identification of the genes for the lactose-specific components of the phosphotransferase system in the lac operon of Staphylococcus aureus.

The nucleotide and deduced amino acid sequences of the lacE and lacF genes, which code for the lactose-specific Enzyme II and Enzyme III of the Staphylococcus aureus phosphotransferase system, are presented. The primary translation products consist of a hydrophobic protein of 572 amino acids (Mr = 62,688) and a polypeptide of 103 amino acids (Mr = 11,372), respectively. The assignment of lacF as the gene for Enzyme IIIlac was based upon the known amino acid sequence of the protein. The identity of lacE as encoding Enzyme IIlac was based upon immunoreactivity of the cloned gene product with antibodies raised against purified Enzyme IIlac from S. aureus and an assay of biological function of the protein expressed in Escherichia coli. The order of the known genes of the S. aureus lac operon is lacF-lacE-lacG, the latter encoding phospho-beta-galactosidase.

Amino Acid Sequence↗
PubMed2007

Secrets of the lac operon. Glucose hysteresis as a mechanism in dietary restriction, aging and disease.

Elevated blood glucose associated with diabetes produces progressive and apparently irreversible damage to many cell types. Conversely, reduction of glucose extends life span in yeast, and dietary restriction reduces blood glucose. Therefore it has been hypothesized that cumulative toxic effects of glucose drive at least some aspects of the aging process and, conversely, that protective effects of dietary restriction are mediated by a reduction in exposure to glucose. The mechanisms mediating cumulative toxic effects of glucose are suggested by two general principles of metabolic processes, illustrated by the lac operon but also observed with glucose-induced gene expression. First, metabolites induce the machinery of their own metabolism. Second, induction of gene expression by metabolites can entail a form of molecular memory called hysteresis. When applied to glucose-regulated gene expression, these two principles suggest a mechanism whereby repetitive exposure to postprandial excursions of glucose leads to an age-related increase in glycolytic capacity (and reduction in beta-oxidation of free fatty acids), which in turn leads to an increased generation of oxidative damage and a decreased capacity to respond to oxidative damage, independent of metabolic rate. According to this mechanism, dietary restriction increases life span and reduces pathology by reducing exposure to glucose and therefore delaying the development of glucose-induced glycolytic capacity.

Aging↗
PubMed1967

Orientation of nonsense codons on the genetic map of the lac operon.

Intracodon recombination is used to orient the nonsense codons UGA, UAG, and UAA on the genetic map of the (lactose) lac operon of Escherichia coli. The 5'-end of these triplets is toward the operator end of the operon. A hypothesis is presented to explain the fact that the frequency of recombination between adjacent nucleotides is many times lower than expected.

Chromosome Mapping↗
PubMed1983

Growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level in Escherichia coli K-12: beta-galactosidase expression in gnd-lac operon fusion strains.

The level of 6-phosphogluconate dehydrogenase is positively correlated with the cellular growth rate. To determine whether growth rate-dependent regulation of expression of gnd, which encodes this enzyme, is carried out by a transcriptional mechanism, the structural genes of the lactose operon were fused to and brought under the control of the gnd promoter through the use of phage Mu d1(Apr lac). Four independent gnd::Mu d1(Apr lac) operon fusion strains were isolated. After the Mu d1 prophage was replaced with lambda p1(209), Lac+ specialized transducing phages carrying the gnd-lac fusions were prepared. These phages were used to demonstrate that the lac genes were fused to the gnd promoter by crossing them with gnd promoter deletion mutants and with polar phage Mu cts-induced gnd mutants. A genetic map of the fusion joints was deduced. The level of beta-galactosidase in each fusion strain was the same in cells growing on acetate as in cells growing on glucose (with specific growth rate constants of 0.1 and 0.55 h-1, respectively) and was unaffected by the presence of a gnd+ gene in trans. Our results suggest that a post-transcriptional mechanism mediates growth rate-dependent regulation of gnd and that this regulation is not autogenous. Models for regulation are discussed with respect to these results and the physiology and DNA sequence of gnd.

DNA, Recombinant↗
PubMed1969

Carabolite repression of the lac operon. Repression of translation.

1. Experiments were devised to show whether catabolite repression of beta-galactosidase synthesis operates at the level of transcription or of translation. Escherichia coli K12 was induced for a short period in non-repressing medium (glycerol-minimal medium), and transcription of the lac operon was terminated by either of two methods; glucose was then added as a source of the catabolite repressor during the subsequent translation of the accumulated beta-galactosidase messenger RNA. 2. When induced bacteria in glycerol medium were infected with T6 phage, which is known to halt transcription, the addition of glucose up to 3min. later diminished the yield of beta-galactosidase. 3. When induced bacteria in glycerol medium were removed from the inducer and resuspended in fresh medium (a process that is also known to halt transcription), the yield of enzyme was again diminished by the presence of glucose in the resuspension medium. 4. It is concluded that repression of beta-galactosidase synthesis can be brought about by the presence of glucose during the translation phase only. 5. In E. coli strain 300U the effect on translation was sufficient to account for almost all the catabolite repression of beta-galactosidase synthesis observed during exponential growth of the organism in glucose-minimal medium. In E. coli strain 200P, however, much more severe repression occurred during exponential growth, and an additional effect of glucose is postulated.

Coliphages↗
PubMed1974

Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: equilibrium aspects.

The regulatory system of the lactose operon has been "modeled" by a set of mass action equations and conservation constraints which describe the system at equilibrium. A "base-set" of values of binding constants and total component concentrations has been assembled from the available experimental data, and the simultaneous equations solved by computer procedures, to yield equilibrium concentrations of all the relevant molecular species. Considering the operator-repressor-inducer system alone, it is shown that the in vivo basal and induced (derepressed) levels of lac enzyme synthesis in both wild-type and certain mutant Escherichia coli can be accounted for only if binding of repressor and repressor-inducer complexes to non-specific DNA sites is included in the calculations as an integral component of the ovrall control system. A similar approach was applied to the RNA polymerase-promoter system to show that sigma factor may modulate the general level of transcription in the cell by "inducing" polymerase off non-specific DNA binding sites, thus making it available to promoters. Competitive and non-competitive models for the interaction of repressor and polymerase at the lac operon can, in principle, be distinguished by these computational procedures, though data sufficient to permit unambiguous differentiation between the models are not available at this time. However, for any competitive binding model the results show that repression in the entire (operator-repressor-RNA polymerase-lac promoter) system can occur only because non-specific binding of the regulatory proteins reduces the concentration of free polymerase, relative to that of repressor, to appropriate levels.

Bacterial Proteins↗
PubMed1967

Transient repression of the lac operon.

Severe transient repression of constitutive or induced beta-galactosidase synthesis occurs upon the addition of glucose to cells of Escherichia coli growing on glycerol, succinic acid, or lactic acid. Only mutants particularily well adapted to growth on glucose exhibit this phenomenon when transferred to a glucose-containing medium. No change in ribonucleic acid (RNA) metabolism was observed during transient repression. We could show that transient repression is pleiotropic, affecting all products of the lac operon. It occurs in a mutant insensitive to catabolite repression. It is established much more rapidly than catabolite repression, and is elicited by glucose analogues that are phosphorylated but not further catabolized by the cell. Thus, transient repression is not a consequence of the exclusion of inducer from the cell, does not require catabolism of the added compound, and does not involve a gross change in RNA metabolism. We conclude that transient repression is distinct from catabolite repression.

Carbon Isotopes↗
PubMed1983

Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions.

The vector Mu d(Apr lac) was utilized to construct operon fusions in the Escherichia coli enterobactin (ent) biosynthetic and transport genes. Enzyme assays indicated a 5- to 15-fold increase in the expression of beta-galactosidase when the fusion strains were grown under iron-deficient conditions. The polarity effects seen by Mu d insertions into entA, entC, and entE were consistent with a single operon, entA(CGB)E. The direction of transcription from iron-regulated promoters was determined by directional transfer of selected genetic markers after the insertion of F'ts114 lac+. Regulatory mutants were isolated in the fusion strains by the selection for constitutive expression of beta-galactosidase and the iron-regulated outer membrane proteins.

Biological Transport↗
PubMed1993

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.

Amino Acid Sequence↗
PubMed1986

Oxygen-regulated stimulons of Salmonella typhimurium identified by Mu d(Ap lac) operon fusions.

Using the technique of Mu d1(Ap lac)-directed lacZ operon fusions, several oxygen-regulated genetic loci were identified in Salmonella typhimurium. Thirteen anaerobically inducible and six aerobically inducible operon fusions were identified. Based on control by the oxrA and oxrB regulatory loci, the anti-lacZ fusions were grouped into three classes: class I loci were regulated by both oxr loci, class II genes were regulated by oxrA only, and class III loci were not affected by either regulatory locus. Several of the anti-lacZ fusions required growth in complex medium before they exhibited the inducible phenotype. While the expression of some of these loci was repressed when organisms were grown in nitrate, others were stimulated by nitrate. Fusions into the hyd and phs loci were identified among the isolated anti-lacZ fusions. Six oxygen-inducible (oxi) operon fusions were also identified. Two of the oxi loci mapped near oxygen-regulatory loci: oxiC near oxrA and oxiE near oxyR. However, neither fusion appeared to occur within the regulatory locus. The data presented serve to further define the aerobic and anaerobic stimulons of S. typhimurium but indicate additional regulatory circuits above those already defined.

Aerobiosis↗
PubMed1974

The location of the repressor binding sites in the lac operon.

The affinity of various mutant lac DNAs for the lac repressor was examined by means of a competition binding assay. The results indicate that there exist at least two binding sites for the lac repressor. The primary binding site is coincidental with the operator, as defined by genetic studies. Mutations of the promoter or the Z gene have little or no effect on the repressor binding properties of this primary binding site, thereby confirming that the operator is distinct from lacP and Z. The secondary binding site(s), which has a 25- to 31-fold lower affinity for the repressor, is located within the operator proximal third of the Z gene.

Binding Sites↗
PubMed1977

Characterization of fusions between the lac operon and the ilv gene cluster in Escherichia coli: ilvC-lac fusions.

By means of the general procedure of Casadaban (J. Mol. Biol. 104: 541-556, 1976), the lac genes carried on a lambda-Mu-1 hybrid phage were inserted into a temperature-inducible Mu-1 prophage that had earlier been inserted into a site near the beginning of the ilvC gene of Escherichia coli strain K-12. Selection of temperature-resistant derivatives of the lysogen resulted in a fusion of the lac genes to a region of deoxyribonucleic acid that is transcribed under the control of the ilvC regulatory elements. A strain bearing the fusion was shown to be inducible for beta-galactosidase by acetohydroxybutyrate, a natural inducer of acetohydroxy acid isomeroreductase. Induction of the lysogen by mitomycin C led to the isolation of a plaque-forming lambda derivative carrying this ilvC-lac fusion.

Bacteriological Techniques↗
PubMed1988

Dual mechanism of repression at a distance in the lac operon.

The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants. Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site. The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression. This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold. Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements. During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously.

Coliphages↗