Inducer and culture medium dependent properties of extracellular laccase from Botrytis cinerea

Both the composition of the culture medium and the nature of the phenolic inducer determine the amount, the rate of formation and the molecular properties of extracellular laccase formed by Botrytis cinerea. Coumaric acid is shown to act as inducer in addition to gallic acid and grape juice. It is suggested that the fungus adapts to different environments by excreting different laccases. These laccases differ in pK, heat stability and substrate specificity but not in K_m values to quinol and oxygen.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction

In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer–repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer–repressor stoichiometry in induction of the lac operon.     

Plant development reprogramming by cynipid gall wasp: proteomic analysis

An insect–plant interaction induced gall formation is where gall wasps change the plant development towards formation of new units to shield and nourish the evolving larvae. The targets of the insect signals and the mechanism of gall development are unknown. To show the molecular pathways that are responsive to the gall wasp, the proteomic approach was used to compare the gall with non-gall plant tissues. We studied three oak gall species (Cynips quercusfolii, Cynips longiventris, and Neuroterus quercusbaccarum) and the host plant (Quercus robur). Among the 21 identified proteins, 18 increased and three decreased in abundance in gall tissue, in comparison to the leaf tissues. Ten proteins were C. quercusfolii responsive, two only with this gall inducer, while seven increased in abundance. Eleven proteins were C. longiventris responsive, and two only with this gall inducer. Sixteen proteins were associated with gall formation by the N. quercusbaccarum and, in this, eight only with this gall inducer. A similar effect on protein abundance occurred as galls in leaf veins (for five proteins). For leaf blades, such a relation was not found. The role of each protein is discussed according to its involvement in the gall formation. Moreover, S-adenosyl methionine synthase, flavone 3-hydroxylase, stress- and pathogenesis-related proteins, and gamma carbonic anhydrase are associated with developmental regulation of plant tissue into a gall.     

Enzymatic production of l-phenylalanine from acetamidocinnamic acid: breeding of an acetamidocinnamate amidohydrolase-hyperproducing mutant

To increase the productivity of l-phenylalanine from acetamidocinnamic acid, we screened bacteria containing high acetamidocinnamate amidohydrolase activity, and strain S-5 containing high activity was isolated from soil. The bacteria were identified as Corynebacterium sp. S-5.When strain S-5 was cultured in a medium containing acetamidocinnamic acid as the sole carbon source or enzyme inducer, the formation of acetamidocinnamate amidohydrolase was observed. This was controlled by catabolite repression. When the strain was cultured in a medium containing glucose and acetamidocinnamic acid as the sole nitrogen source, it showed low acetamidocinnamate amidohydrolase activity and an increased doubling time.To obtain acetamidocinnamate amidohydrolase-hyperproducing strain, we enriched cells growing faster than strain S-5 in a medium containing glucose and acetamidocinnamic acid by continuous culture of mutagenized cells. Mutant C-23 had 12-fold the enzyme production and 3-fold the growth rate of the wild-type strain in a medium containing glucose. Acetamidocinnamate amidohydrolase formation in the mutant did not require acetamidocinnamic acid as enzyme inducer and was resistant to catabolite repression.     

Half-of-the sites reactivity in the catalytic mechanism of yeast glyceraldehyde 3-phosphate dehydrogenase

Novick &; Weiner (1957) proposed a model in which induction of the lac operon with suboptimal concentrations of inducer generates a population containing both uninduced and fully induced cells. The latter arise as cells acquire the galactoside transport system, thus initiating an autocatalytic cycle of induction since this permease can transport an inducer for its own synthesis. Evidence in favor of this model has been obtained from direct measurements of the enzyme content of individual cells, using a fluorogenic assay sensitive to one molecule of β-d-galactosidase. Fully induced cells, at the predicted frequency, were found in suboptimally induced populations of wild type strains, and of a strain lacking thiogalactoside transacetylase, but not of a strain lacking galactoside permease. In the wild type, the frequency of cells with an enzyme content intermediate between uninduced and fully induced levels was greater than the frequency predicted for cells within the autocatalytic cycle of induction. According to the model, then, in some of these cells, induction of β-d-galactosidase has occurred without formation of the permease necessary to initiate accumulation of inducer.     

lac repressor-operator interaction. Analysis of the X86 repressor mutant

In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator sites on Escherichia coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli DNA. In a partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to either non-operator sites on E. coli DNA or to an O^c operator, the formation of complexes between X86 repressor and wild-type operator is favored by low inducer concentrations. Repression of the lac enzymes increases drastically in the X86 mutant in the absence of DNA synthesis in vivo. A new explanation for the in vivo characteristics of the X86 mutant is suggested.     

The intracellular galactoglycome in Trichoderma reesei during growth on lactose

Lactose (1,4-0-β-d-galactopyranosyl-d-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for—among other purposes—use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography–mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-β-1,6-Gal-β-1,4-Glc/Fru and Gal-β-1,4-Gal-β-1,4-Glc/Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-β-1,3/1,4/1,6-Gal; Gal-β-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-β-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-β-1,4-Gal-β-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation.     

Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii

The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni²⁺-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.     

Regulatory aspects of endoglucanase production by the brown-rot fungusGloeophyllum trabeum

Regulation of endoglucanase formation by the brown-rot fungusGloeophyllum trabeum was investigated. This fungus produced endoglucanases in the presence of monosaccharides such as glucose or mannose as the sole carbon source, but the expression of these enzymes was four to five times higher in the presence of cellulose or cellobiose. In a lactose- or glucose-containing medium, endoglucanase production was induced by cellobiose. Glucose and glycerol did not repress enzyme production. We concluded that endoglucanase production by brown-rot fungi is inducible by cellulose and not subject to catabolite repression. Cellobiose is the most effective inducer of the system.     

Anatomical and histochemical characterization of in vitro haustorium from roots of Castilleja tenuiflora

In vitro induction of haustoria from Castilleja tenuiflora Benth. was achieved by applying 25 μM catechin, 25 μM vanillin, or 25 μM H₂O₂. Of the treatments tested, 25 μM vanillin was the strongest inducer of haustoria in C. tenuiflora roots in vitro (up to 3 haustoria per root). Haustorium development occurred laterally and was observable 14 d after inducer application. It was characterized by elongation of the epidermal cells and division of the inner cortical cells which also possessed abundant nuclei. Histochemical analysis using 3,3-diaminobenzidine (DAB) and diphenylboric acid 2-aminoethyl ester (DBPA) indicated that the formation of haustoria was associated with the accumulation of H₂O₂ and flavonoids.     

A novel mutant of the lac transport system of Escherichia coli.

A new type of lactose permease mutant, called lacY^f, does not actively transport the usual substrates; but it does facilitate the entry of β-galactosides into Escherichia coli K-12. The kinetics of facilitated entry, as assayed by hydrolysis of o-nitrophenyl-β-d-galactopyranoside by intact cells are identical to those observed with wild-type permease. However, the mutant permease activity is not affected by SH reagents or the substrate analog β-d-galactosyl-1-thio-β-d-galactopyranoside which strongly inhibit wild-type activity. Furthermore, the kinetics of formation of permease in the mutant following addition of inducer and the kinetics subsequent to removal of inducer differ strikingly from those observed in wild-type strains. The results are consistent with a block in the maturation of permease in the mutant resulting in the accumulation of a large amount of permease precursor. Studies of the $\text{lacY}{}^{\mathrm{+}}\text{lacY}{}^{\mathrm{f}}$ heterogenotes provide evidence for a subunit structure for the lactose permease.     

Two Systems for Conditional Gene Expression in Myxococcus xanthus Inducible by Isopropyl-β-d-Thiogalactopyranoside or Vanillate

Conditional expression of a gene is a powerful tool to study its function and is typically achieved by placing the gene under the control of an inducible promoter. There is, however, a dearth of such inducible systems in Myxococcus xanthus, a well-studied prokaryotic model for multicellular development, cell differentiation, motility, and light response and a promising source of secondary metabolites. The few available systems have limitations, and exogenously based ones are unavailable. Here, we describe two new, versatile inducible systems for conditional expression of genes in M. xanthus. One employs isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer and is inspired by those successfully applied in some other bacteria. The other requires vanillate as an inducer and is based on the system developed originally for Caulobacter crescentus and recently adapted for mammalian cells. Both systems are robust, with essentially no expression in the absence of an inducer. Depending on the inducer and the amounts added, expression levels can be modulated such that either system can conditionally express genes, including ones that are essential and are required at high levels such as ftsZ. The two systems operate during vegetative growth as well as during M. xanthus development. Moreover, they can be used to simultaneously induce expression of distinct genes within the same cell. The conditional expression systems we describe substantially expand the genetic tool kit available for studying M. xanthus gene function and cellular biology.     

Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures

Background

Escherichia coli (E. coli) is the most abundant expression host for recombinant proteins. The production efficiency is dependent on a multitude of parameters. Therefore, high-throughput applications have become an increasingly frequent technique to investigate the main factors. Within this study, the effects of temperature, induction time and inducer concentration on the metabolic state and the product formation were extensively examined. Induction profiling of E. coli Tuner(DE3) pRhotHi-2-EcFbFP was performed in 48-well Flowerplates and standard 96-well plates using a robotic platform. In parallel shake flask cultivations, the respiration activity of the microorganisms was analyzed. Therefore, two online-monitoring systems were applied: the BioLector for microtiter plates and the RAMOS-device for shake flasks. The impact of different induction conditions on biomass and product formation as well as on the oxygen transfer rate was surveyed.

Results

Different optimal induction conditions were obtained for temperatures of 28, 30, 34, and 37 °C. The best inducer concentrations were determined to be between 0.05 and 0.1 mM IPTG for all investigated temperatures. This is 10–20 times lower than conventional guidelines suggest. The induction time was less relevant when the correct inducer concentration was chosen. Furthermore, there was a stronger impact on growth and respiration activity at higher temperatures. This indicated a higher metabolic burden. Therefore, lower IPTG concentrations were advantageous at elevated temperatures. Very similar results were obtained in standard 96-well plates.

Conclusion

Two online-monitoring systems were successfully used to investigate the optimal induction conditions for the E. coli Tuner(DE3) pRhotHi-2-EcFbFP strain (lacY deletion mutant) at four different temperatures. The experimental effort was reduced to a minimum by integrating a liquid handling robot. To reach the maximum product formation, a detailed induction analysis was necessary. Whenever the cultivation temperature was changed, the induction conditions have to be adapted. Due to the experimental options provided by the BioLector technology, it was found that the higher the cultivation temperature, the lower the inducer concentration that has to be applied.

     

A novel role for enzyme I of the Vibrio cholerae phosphoenolpyruvate phosphotransferase system in regulation of growth in a biofilm

Glucose is a universal energy source and a potent inducer of surface colonization for many microbial species. Highly efficient sugar assimilation pathways ensure successful competition for this preferred carbon source. One such pathway is the phosphoenolpyruvate phosphotransferase system (PTS), a multicomponent sugar transport system that phosphorylates the sugar as it enters the cell. Components required for transport of glucose through the PTS include enzyme I, histidine protein, enzyme IIA^Glc, and enzyme IIBC^Glc. In Escherichia coli, components of the PTS fulfill many regulatory roles, including regulation of nutrient scavenging and catabolism, chemotaxis, glycogen utilization, catabolite repression, and inducer exclusion. We previously observed that genes encoding the components of the Vibrio cholerae PTS were coregulated with the vps genes, which are required for synthesis of the biofilm matrix exopolysaccharide. In this work, we identify the PTS components required for transport of glucose and investigate the role of each of these components in regulation of biofilm formation. Our results establish a novel role for the phosphorylated form of enzyme I in specific regulation of biofilm-associated growth. As the PTS is highly conserved among bacteria, the enzyme I regulatory pathway may be relevant to a number of biofilm-based infections.     

The effects of alkylhydroxybenzenes on homoserine lactone-induced manifestations of quorum sensing in bacteria

The effects of four alkylhydroxybenzene (AHB) homologs with different hydrocarbon chain lengths on the synthesis of violacein pigment induced by C₆-homoserine lactone (HSL) and biofilm formation by Chromobacterium violaceum NCTC 13274 and on Escherichia coli pAL103 bioluminescence in the presence of C₆-oxo-HSL were studied. Alkylhydroxybenzenes inhibit the growth of C. violaceum increased in the C₅-AHB → C₁₂-AHB series in the absence of this activity in C₁-AHB. Subinhibitory AHB concentrations reduced violacein production and suppressed biofilm formation. These effects were presented as individual and group regression dependencies between the analysed parameters. Using the bioluminescent model, the regulatory effects of AHBs were not associated with their direct competition with HSL and that they develop as a result of changes in the sensitivity of bacterial cells to the respective quorum sensing inducer.     

Induction of Lipomyces starkeyi Dextranase

Lipomyces starkeyi ATCC 20825 is a derepressed mutant derived from L. starkeyi ATCC 12659. It requires the presence of an inducer before it produces dextranase. This study was undertaken to determine the most efficient, commercially feasible method for inducing this enzyme. The following compounds induced dextranase synthesis: 1-O-β-methyl-glucopyranoside, 1-O-α-methyl-glucopyranoside, dextran, isomaltopentose, isomaltotetraose, isomaltotriose, and isomaltose. 1-O-β-Methyl-glucopyranoside was found to be a gratuitous inducer. Early in the growth phase, cells produced higher specific levels of enzyme than they did in late log phase. The length of exposure of the yeast cells to the inducer also affected the amount of dextranase produced. The maximum amount of enzyme was produced after 12 h of exposure to the inducer. The saturation concentration was the same for all inducers tested, i.e., approximately 1 mg of inducer for every 2 × 10⁸ cells.     

Microsomal oxidation of bromo-, chloro- and fluorobiphenyls

1. The metabolism of 2-, 3-, 4-bromo-, 2-, 4-chloro-, and 2-fluorobiphenyl by hepatic microsomes isolated from control and Aroclor 1254-treated rats and pigeons was studied.2. Meta and para as well as dihydroxylated metabolites were detected, but para hydroxylation was the preferred route of metabolism with all of the substrates used.3. The overall rates of hydroxylation were greater with hepatic microsomes from rats than from pigeons.4. Treatment with Aroclor 1254, a potent inducer of hepatic monooygenases, resulted in increased rates of metabolism and in the enhanced formation of diol metabolites. Metabolism of halobiphenyls by induced P450 isoenzymes altered the regioselective hydroxylation pathways.5. Ortho- and meta halosubstituted biphenyls were less rapidly metabolised when compared with paru substituted isomers.     

Effect of toluidine blue on pollen germination and pollen tube growth

Toluidine blue is known to induce gynogenic haploids in significant numbersin Populus]. Because the efficacy of a chemical in inducing gynogenesis depends largely on its effeot on pollen germination, on pollen tube growth, and on male gamete formation, the effect of toluidine blue (0, 1, 10 and 100 mgl-1) on these processes was studied in treated pistils ofSolatium nigrum (4 X), as well as on cultured pollen grains ofS. nigrum andTrigonella foenumgraecum. Irrespective of the time of application, toluidine blue (1 and 10 mg I-1) had no effect on pollen germination or pollen tube growth in pistils ofS. nigrum; at 100 mg I-1 it invariably inhibited both the processes. Almost similar responses were elicited by cultured pollen grains. InT. foenum-graecum toluidine blue had no effeot on pollen germination and suppressed tube growth. Gamete formation was inhibited, to various degrees, at all the concentrations tested; at 100 ing I-1 hardly any pollen tube showed gamete formation. Based on our results, and those on other systems, the potentiality of toluidine blue as an inducer of gynogenesis has been analysed.