Polycistronic Effects of Catabolite Repression on the lac Operon

The catabolite repression caused by glucose and glucose-6-phosphate has been studied for both beta-galactosidase and thiogalactoside transacetylase, the products of the operator proximal and distal cistrons of the lac operon, respectively. We find that both cistrons are affected coordinately by this form of repression. We also find that a single alteration at the lac promoter region is sufficient to abolish sensitivity to repression of both cistrons. From this, we conclude that there is only one target site for catabolite repression in the lac operon.     

Regulation of lac Operon Expression: Reappraisal of the Theory of Catabolite Repression

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.     

Origin of bistability in the lac Operon

Multistability is an emergent dynamic property that has been invoked to explain multiple coexisting biological states. In this work, we investigate the origin of bistability in the lac operon. To do this, we develop a mathematical model for the regulatory pathway in this system and compare the model predictions with other experimental results in which a nonmetabolizable inducer was employed. We investigate the effect of lactose metabolism using this model, and show that it greatly modifies the bistable region in the external lactose (Le) versus external glucose (Ge) parameter space. The model also predicts that lactose metabolism can cause bistability to disappear for very low Ge. We have also carried out stochastic numerical simulations of the model for several values of Ge and Le. Our results indicate that bistability can help guarantee that Escherichia coli consumes glucose and lactose in the most efficient possible way. Namely, the lac operon is induced only when there is almost no glucose in the growing medium, but if Le is high, the operon induction level increases abruptly when the levels of glucose in the environment decrease to very low values. We demonstrate that this behavior could not be obtained without bistability if the stability of the induced and uninduced states is to be preserved. Finally, we point out that the present methods and results may be useful to study the emergence of multistability in biological systems other than the lac operon.     

Repression of the Histidine Operon: Effect of the First Enzyme on the Kinetics of Repression

Kinetic studies on repression of the enzymes for histidine biosynthesis in Salmonella typhimurium showed that, upon addition of histidine to a derepressed culture, the enzymes became repressed in a temporal sequence which corresponds with the positional sequence of the genes in the histidine operon. This serial pattern of repression occurred under conditions in which the feedback site of the first enzyme for histidine biosynthesis is intact. When this site was rendered nonfunctional the pattern of repression was changed so that all of the enzymes became repressed concomitantly. These results suggest that the first enzyme for histidine biosynthesis plays a hitherto unrecognized role in control of the histidine system.     

Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.     

Transient repression of the lac operon - the effect of a lac promoter deletion

Experiments have been done to show whether the lac promoter delection L1, which partly alleviates catabolite repression, also affects transient repression of lac. In stain L1/F'M15 all of the beta-galactosidase is synthesized from a chromosomal gene cis to L1, whereas 98% of the thiogalactosidase transacetylase is synthesized from an episomal gene cis to an intact i-p-o region. The addition of glucose to induced cultures of strain L1/F'M15 growing in glycerol medium caused extensive transient repression of transacetylase but almost no transient repression of beta-galactosidase. In control experiments with a diploid stain of genotype p(+)z(+)a(-)/F'p(+)z(-)a(+) the two enzymes suffered equal transient repression. Thus L1 substantially relieves transient repression.     

Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.     

利用Red重组工程技术解决外源基因在大肠杆菌染色体lac操纵子中的表达

为了应用Red重组工程技术实现外源基因在大肠杆菌染色体上的表达, 寻找染色体上外源蛋白的稳定高效表达位点, 使用Red重组工程系统和kan/sacB无痕迹修饰技术, 将易于定量分析的荧光素酶报告基因替换DY330染色体lac操纵子中的lacZ基因。检测该位点的表达效率结果显示: 大肠杆菌染色体上lac操纵子能够高效稳定表达外源基因, 初步证明了染色体可以作为外源蛋白或抗原的表达载体, 不会影响细菌的生长繁殖。     

利用Red重组工程技术解决外源基因在大肠杆菌染色体lac操纵子中的表达

为了应用Red重组工程技术实现外源基因在大肠杆菌染色体上的表达, 寻找染色体上外源蛋白的稳定高效表达位点, 使用Red重组工程系统和kan/sacB无痕迹修饰技术, 将易于定量分析的荧光素酶报告基因替换DY330染色体lac操纵子中的lacZ基因。检测该位点的表达效率结果显示: 大肠杆菌染色体上lac操纵子能够高效稳定表达外源基因, 初步证明了染色体可以作为外源蛋白或抗原的表达载体, 不会影响细菌的生长繁殖。     

Cyclic 3′,5′-Adenosine Monophosphate and N-Acetyl-glucosamine-6-Phosphate as Regulatory Signals in Catabolite Repression of the lac Operon in Escherichia coli

When an Escherichia coli mutant lacking the enzyme N-acetyl-glucosamine-6-phosphate (AcGN6P) deacetylase is grown in a succinate-mineral salts medium and exposed to an exogenous source of N-acetylglucosamine, approximately 20 to 30 pmoles of AcGN6P per mug of cell dry weight will accumulate in these cells. This accumulation occurs within 2 to 4 min after the addition of N-acetylglucosamine and is coincident with the production of a severe permanent catabolite repression of beta-galactosidase synthesis. This repression does not occur if adenosine 3',5'-cyclic phosphate (cyclic AMP) is added to the cells before AcGN6P accumulates. An immediate derepression occurs when cyclic AMP is added to cells that have already accumulated a large AcGN6P pool. These findings are consistent with the view that low-molecular-weight carbohydrate metabolites and cyclic AMP play key roles in the catabolite repression phenomenon, and that metabolites such as AcGN6P may participate in the represion mechanism by influencing either the formation or degradation of cyclic AMP in E. coli.     

pBR322-Red在大肠杆菌lac操纵子基因敲除和敲入中的应用

pBR322-Red是一种新型重组工程系统,它携带了λ-噬菌体Red重组酶基因和一系列调控元件.对pBR322-Red最优重组条件进行探索后应用该质粒提供的体内同源重组功能,在菌株W3110体内,对染色体上的lac操纵子进行了基因修饰,包括:①运用kan/sacB选择反选择方法和重叠引物方法敲除了阻遏基因lacⅠ,②运用kan/sacB选择反选择方法和线性双链DNA介导的DNA重组方法将报告基因lacZ敲入lacA和lacY的位置,并且首次测定了报告基因lacZ在这三个结构基因位置的组成性表达情况.结果表明运用不同的重组策略,pBR322-Red系统都能方便有效地对大肠杆菌W3110染色体进行基因敲除和敲入修饰.     

Evidence for an Altered Operator Specificity: Catabolite Repression Control of the Leucine Operon in Salmonella typhimurium

A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.     

Hypersensitivity to Catabolite Repression in the l-Arabinose Operon of Escherichia coli B/r Is trans Acting

Mutations causing hypersensitivity to catabolite repression have been assigned to gene araC (activator protein) by complementation analysis. The araO (operator region) is non-essential for catabolite repression.     

Physiological Basis of Transient Repression of Catabolic Enzymes in Escherichia coli

Transient repression of catabolic enzymes occurs in cells that encounter a new carbon compound in their growth medium, but only when the cells contain the enzyme catalyzing the transfer of phosphate from phosphoenolpyruvate to a small heat-stable protein (HPr), as well as a permease capable of transporting the new compound across the cell membrane. The newly added compound need not be metabolized. The degree and duration of the transient repression have no obvious relation to the intracellular level of the exogenously added compound. It is suggested that the actual passage of the compound through the cell membrane is responsible for the repression.