Binding of polynucleotides to fibroblasts. Effects of complex formation with vinyl analogs of nucleic acids

The nitrocellulose filter assay was used to study the effect of the DNA denaturants glycerol and dimethylsulfoxide (Me2SO) on the lac repressor-operator interaction. Both glycerol and Me2SO decrease the rate of dissociation (kb) of the repressor-operator complex but do not significantly alter the rate of association of repressor and operator. In the presence of 10% Me2SO an almost 10-fold increase of affinity of repressor for operator is observed. A small increase in affinity of repressor for Escherichia coli DNA, chicken blood DNA, and poly(dA-dT) is also found. The results lead to the conclusion that lac repressor when interacting with the operator causes local destabilization of the DNA.     

Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding.

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.     

Transposon-mediated adaptive and directed mutations and their potential evolutionary benefits

Transposons, mobile genetic elements that can hop from one chromosomal location to another, are known to be both beneficial and deleterious to the cell that bears them. Their value in accelerating evolutionary adaptation is well recognized. We herein summarize published research dealing with these elements and then move on to review our own research efforts which focus on a small transposon that can induce mutations under the control of host factors in a process that phenotypically and mechanistically conforms to the definition of 'directed mutation'. Directed mutations occur at higher frequencies when they are beneficial, being induced by the stress condition that they relieve. Here, we review evidence for transposon-mediated directed mutation in Escherichia coli. Deletion mutants in the crp gene can not grow on glycerol (Glp(-)); however, these cells mutate specifically to efficient glycerol utilization (Glp(+)) at rates that are greatly enhanced by the presence of glycerol or the loss of the glycerol repressor (GlpR). These rates are greatly depressed by glucose or by glpR overexpression. Of the four tandem GlpR-binding sites (O1-O4) in the control region of the glpFK operon, O4 (downstream) specifically controls glpFK expression while O1 (upstream) controls mutation rate. Mutation is due to insertion of the small transposon IS5 into a specific site just upstream of the glpFK promoter. Mutational control by the glycerol regulon repressor GlpR is independent of the selection and assay procedures, and IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish the principle of transposon-mediated directed mutation, identify a protein responsible for its regulation, and define essential aspects of the mechanism.     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Catabolite repression of inositol dehydrogenase and gluconate kinase syntheses in Bacillus subtilis

The regulation of induction of inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) was studied in Bacillus subtilis. Inositol dehydrogenase is induced by myo-inositol and gluconate kinase is induced by D-gluconate. Both inductions were strongly repressed by rapidly metabolizable carbohydrates such as D-glucose, D-mannose, D-fructose and glycerol (D-glucose had the strongest repressive effect) but they were weakly repressed by slowly metabolizable carbohydrates. Although each carbohydrate exerted a stronger effect on the induction of inositol dehydrogenase than that of gluconate kinase, it showed a similar tendency with respect to the degree of repression of each induction. This catabolite repression could not be diminished by addition of cyclic AMP to medium. In addition, non-metabolizable D-glucose analogues had no or weak repressive effects. On the assumption that rapidly metabolizable carbohydrates might be metabolized to repress both inductions, it was investigated whether several mutants blocked in the Embden-Meyerhof pathway could produce metabolite(s) (repressor) to repress them. A phosphoglycerate kinase (EC 2.7.2.3) deficient mutant could produce the repressor from D-glucose, D-mannose, D-fructose and glycerol but other mutants could not produce it from carbohydrates unable to be metabolized ineach mutant. Thus, catabolite repression of both enzyme inductions seemed to be under similar regulation. The identification of the possible repressor of the induction of inositol dehydrogenase and gluconate kinase in vivo was discussed.     

A mechanism of transposon-mediated directed mutation

Directed mutation is a proposed process that allows mutations to occur at higher frequencies when they are beneficial. Until now, the existence of such a process has been controversial. Here we describe a novel mechanism of directed mutation mediated by the transposon, IS 5 in Escherichia coli . crp deletion mutants mutate specifically to glycerol utilization (Glp⁺) at rates that are enhanced by glycerol or the loss of the glycerol repressor (GlpR), depressed by glucose or glpR overexpression, and RecA-independent. Of the four tandem GlpR binding sites ( O1–O4 ) upstream of the glpFK operon, O4 specifically controls glpFK expression while O1 primarily controls mutation rate in a process mediated by IS 5 hopping to a specific site on the E. coli chromosome upstream of the glpFK promoter. IS 5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish an example of transposon-mediated directed mutation, identify the protein responsible and define the mechanism involved.     

Galactose repression of beta-galactosidase induction in Escherichia coli

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.     

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol‐glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol‐glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04–0.06 h^?1, while ROL yield decreased along the whole dilution rate range evaluated (0.03–0.1 h^?1). Compared to production level achieved with methanol‐only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g‐biomass^?1 h^?1), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut⁺ phenotype for heterologous protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:707–714, 2015     

Regulation of formation of the intracellular beta-galactosidase activity of Aspergillus nidulans

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.     

Scalable method to determine mutations that occur during adaptive evolution of Escherichia coli

Denaturing HPLC was used to determine mutations occurring during the adaptive evolution of Escherichia coli K-12. The strains were evolved over 700 generations on glycerol as the sole carbon source from a sub-optimal to an optimal growth rate. The mutations detected by direct sequencing of amplicons of the glycerol-phosphate regulon repressor (glpR) gene were a synonymous substitution Val20Val in two separately evolved strains. Non-synonymous substitutions, Val119Gly and Gly179Trp, were also observed in each of the two strains. This procedure can be scaled to determine genome-scale sequence variations that have occurred during adaptive evolution.     

Three Kinds of Controls Affecting the Expression of the glp Regulon in Escherichia coli

Three kinds of control mechanisms govern the expression of the members of the glp regulon for glycerol and sn-glycerol 3-phosphate (G3P) catabolism in Escherichia coli K-12: specific repression by the product of the glpR gene; catabolite repression; and respiratory repression (the effect exerted by exogenous hydrogen acceptors). The operons of the glp system show different patterns of response to each control. By growing in parallel a mutant strain with temperature-sensitive repressor (glpR(ts)) and an isogenic control with a deletion in the regulator gene at progressively higher temperatures, it was possible to show that the synthesis of aerobic G3P dehydrogenase (glpD product) is far more sensitive to specific repression than that of either glycerol kinase (glpK product) or G3P transport (glpT product). Conversely, in the strain with a deletion in the regulator gene, the syntheses of glycerol kinase and G3P transport are more sensitive to catabolite repression than that of the aerobic G3P dehydrogenase. The levels of the two flavoprotein G3P dehydrogenases vary in opposite directions in response to changes of exogenous hydrogen acceptors. For example, the ratio of the aerobic enzyme to the anaerobic enzyme (specified by glpA) is high when molecular oxygen or nitrate serves as the hydrogen acceptor and low when fumarate plays this role. This trend is not influenced by the addition of cyclic adenosine 3',5'-monophosphate to the growth medium. Thus, respiratory repression most likely involves a third mechanism of control, independent of specific or catabolite repression.     

Catabolite repression of the Bacillus subtilis xyl operon involves a cis element functional in the context of an unrelated sequence, and glucose exerts additional xylR-dependent repression.

Catabolite repression (CR) of xylose utilization by Bacillus subtilis involves a 14-bp cis-acting element (CRE) located in the translated region of the gene encoding xylose isomerase (xylA). Mutations of CRE making it more similar to a previously proposed consensus element lead to increased CR exerted by glucose, fructose, and glycerol. Fusion of CRE to an unrelated, constitutive promoter confers CR to beta-galactosidase expression directed by that promoter. This result demonstrates that CRE can function independently of sequence context and suggests that it is indeed a generally active cis element for CR. In contrast to the other carbon sources studied here, glucose leads to an additional repression of xylA expression, which is independent of CRE and is not found when CRE is fused to the unrelated promoter. This repression requires a functional xylR encoding Xyl repressor and is dependent on the concentrations of glucose and the inducer xylose in the culture broth. Potential mechanisms for this glucose-specific repression are discussed.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

Circular dichroism studies of the binding of o-nitrophenyl-beta-D-fucoside and o-nitrophenyl-beta-D-galactoside to lac repressor.

The binding of o-nitrophenyl-beta-D-fucoside and o-nitrophenyl-beta-D-galactoside to Escherichia coli lac repressor was investigated by circular dichroism in the wavelength range 300--400 nm corresponding to the o-nitrophenyl chromophores. The CD signal of both ligands drastically changed when they bound to lac repressor due to the asymmetric interaction of the o-nitrophenyl ring with chemical groups of protein. The CD spectra of bound ligands indicate close similarity between the environment of o-nitrophenyl-beta-D-fucoside and o-nitrophenyl-beta-D-galactoside on lac repressor. The CD signal is used to calculate the binding parameters (K and n) to lac repressor. It is demonstrated that the limited proteolytic digestion of lac repressor which gives a 'core protein' does not affect the environment of both ligands on the protein.     

How Lac repressor finds lac operator in vitro.

Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.     

Repressor and Antirepressor in the Regulation of Staphylococcal Penicillinase Synthesis

5-methyltryptophan (5MT) induces penicillinase synthesis in Staphylococcus aureus. The analog is incorporated into protein by both wild-type and tryptophan-starved cells. Since normal penicillinase repressor appears to contain tryptophan even though penicillinase itself does not, it is concluded that 5MT induces penicillinase synthesis by becoming incorporated into the penicillinase repressor and thereby inactivating the repressor. Thus biochemical data support the existence of a penicillinase repressor and indicate that penicillinase synthesis is regulated by negative control and not by positive control.-In the absence of exogenous tryptophan, staphylococcal penicillinase induction can be inhibited by 7-azatryptophan (7azaT). Because 7azaT is incorporated into protein by tryptophan-starved cells, it is concluded that 7azaT blocks penicillinase induction by inactivating a penicillinase regulatory protein into which the analog has been incorporated. Incorporation of 7azaT does not appear to inactivate the operator binding site or the effector binding site on the penicillinase repressor. Therefore, it appears that 7azaT blocks penicillinase induction by inactivating the penicillinase antirepressor, a protein required for inactivation of the penicillinase repressor and, hence, required for penicillinase induction.