cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP) 2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP) 4 complex alters the surroundings of Trp85. Timeresolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP) 2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP) 2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 Å in apo-CRP to 18.7 Å in the CRP-(cAMP) 2 complex. The binding of cAMP in the CRP-(cAMP) 4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to % 20 Å . The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.Keywords: allosteric regulation; cAMP receptor protein; emission anisotropy; Escherichia coli; fluorescence. cAMP receptor protein (CRP), which is allosterically activated by cAMP, regulates transcription of over 100 genes in Escherichia coli [1,2]. Upon binding the cyclic nucleotide, CRP undergoes an allosteric conformational change that allows it to bind specific DNA sequences with increased affinity [3]. CRP is a dimeric protein, composed of two identical 209-amino-acid subunits. Each subunit of CRP has a molecular mass of 23.6 kDa, as deduced from the amino-acid sequence. Individual subunits fold into two domains [4]. The larger N-terminal domain (residues 1-133) is responsible for dimerization of CRP and for interaction with the allosteric effector, cAMP. The smaller C-terminal domain (residues 139-209) is responsible for interaction with DNA through a helix-turn-helix motif. CRP recognizes a 22-bp, symmetric DNA site [5]. Amino-acid residues 134-138 form a flexible hinge which covalently couples two domains. Recent studies of the crystal structure of the CRP-DNA complex showed that each protein subunit binds two cAMP molecules with different affinities [6]. Higher-affinity sites, where the nucleotide binds in the anti conformation, are buried within the N-terminal domains, whereas loweraffinity binding sites (where the bound cAMP has a syn conformation) are located at the interface formed by the two C-termin...

The aim of this study was to determine the influence of two different Wickerhamomyces anomalus strains, CBS 1982 and CBS 5759, on the chemical composition and sensory characteristics of Gloster apple wines. They were inoculated into unpasteurized as well as pasteurized apple musts together with a S. cerevisiae strain as a mixed culture. Fermentation kinetics, basic enological parameters, antioxidant properties as well as selected polyphenol, volatile compound, and organic acid contents were analyzed during the experiments. Apple wines obtained after spontaneous fermentation were characterized by high volatile acidity, increased concentrations of acetaldehyde, and volatile esters, as well as the lowest amounts of ethyl alcohol and higher alcohols compared with other samples. Addition of 0.05 g L(-1) W. anomalus killer strains to the unpasteurized must significantly changed the fermentation kinetics and chemical composition of apple wines. The value of volatile acidity was highly decreased, while the amount of higher alcohols and titratable acidity increased. Pasteurization of must improved the fermentation efficiency. Higher amounts of polyphenol compounds and lower amounts of malic acid were also detected. Application of W. anomalus strains together with S. cerevisiae yeast as a mixed culture positively influenced the chemical composition and sensory features of produced apple wines.

Phytate content, bioavailability and myo-inositol phosphates profile were analysed in pseudocereals (i.e., white and coloured quinoa, buckwheat) subjected to thermal processing and solid-state fermentation with Rhizopus oligosporus. Fair buckwheat groats were characterised by the highest level of phytate (18.3 µmol/g), with 54% share of InsP 6 fraction. Processing (cooking, roasting, tempe-type fermentation) resulted in the significant decrease in phytate content, its bioavailability and the share of higher phosphorylated forms of myo-inositol (InsP 6-5) in the profile of all the samples. Fermentation of cereals resulted in generation of high amounts of inositol triphosphates, particularly in the case of buckwheat (32% share in the whole profile). The path of dephosphorylation, identified after fermentation of quinoa seeds including intermediate products-Ins(1,2,4,5,6)P 5 and Ins(1,2,6)P 3confirmed that the dominant phytase activity could be attributed to the presence of microbial 3-phytases from R. oligosporous.

Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, theta1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, theta2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of theta1 to the value of 30.7 ns, whereas theta2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, theta1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.

A new method was proposed to produce alginate bio-films containing Pichia membranifaciens and Wickerhamomyces anomalus killer yeast to control the post-harvest fungal decay in organic apples caused by Botrytis cinerea and Penicillium italicum. Coatings with W. anomalus killer yeast effectively controlled the growth of P. italicum during storage at 22 °C. W. anomalus killer yeast incorporated in alginate reduced the P. italicum incidence from 90% (control) to 35% after 14 days of storage at 22 °C. Alginate biofilms with W. anomalus or P. membranifaciens also limited the incidence of the fungal decay of apples inoculated with B. cinerea compared with the control fruits, although the antagonistic capability against B. cinerea was lower than against P. italicum. The survival of W. anomalus cells in alginate coating was higher than P. membranifaciens. The incorporation of killer yeasts into alginate had no significant effect on the mechanical properties (tensile strength, percent elongation at break) of alginate coating, however, they increased the thickness of the biofilm. The bioactive coating reduced the fruit weight loss and had no significant effects on the fruit firmness during storage at 2 °C. As organic apples, produced without any synthetic fungicides, are especially prone to fungal decay during storage, the proposed alginate biofilms containing killer yeast seem to be a very promising solution by offering non-chemical, biological control of post-harvest pathogens.