Side-stream products of malting: a neglected source of phytochemicals.

Whole grain consumption reduces the risk of several chronic diseases. A major contributor to the effect is the synergistic and additive effect of phytochemicals. Malting is an important technological method to process whole grains; the main product, malted grain, is used mainly for brewing, but the process also yields high amounts of side-stream products, such as rootlet. In this study, we comprehensively determined the phytochemical profile of barley, oats, rye, and wheat in different stages of malting and the subsequent extraction phases to assess the potential of malted products and side-streams as a dietary source of bioactive compounds. Utilizing semi-quantitative LC-MS metabolomics, we annotated 285 phytochemicals from the samples, belonging to more than 13 chemical classes. Malting significantly altered the levels of the compounds, many of which were highly increased in the rootlet. Whole grain cereals and the malting products were found to be a diverse and rich source of phytochemicals, highlighting the value of these whole foods as a staple. The characterization of phytochemicals from the 24 different sample types revealed previously unknown existence of some of the compound classes in certain species. The rootlet deserves more attention in human nutrition, rather than its current use mainly as feed, to benefit from its high content of bioactive components.

Acidipropionibacterium virtanenii sp. nov., isolated from malted barley.

A Gram-stain-positive, catalase-positive and pleomorphic rod organism was isolated from malted barley in Finland, classified initially by partial 16S rRNA gene sequencing and originally deposited in the VTT Culture Collection as a strain of Propionibacterium acidipropionici (currently Acidipropionibacterium acidipropionici). The subsequent comparison of the whole 16S rRNA gene with other representatives of the genus Acidipropionibacterium revealed that the strain belongs to a novel species, most closely related to Acidipropionibacterium microaerophilum and Acidipropionibacterium acidipropionici, with similarity values of 98.46 and 98.31 %, respectively. The whole genome sequencing using PacBio RS II platform allowed further comparison of the genome with all of the other DNA sequences available for the type strains of the Acidipropionibacterium species. Those comparisons revealed the highest similarity of strain JS278T to A. acidipropionici, which was confirmed by the average nucleotide identity analysis. The genome of strain JS278T is intermediate in size compared to the A. acidipropionici and Acidipropionibacterium jensenii at 3 432 872 bp, the G+C content is 68.4 mol%. The strain fermented a wide range of carbon sources, and produced propionic acid as the major fermentation product. Besides its poor ability to grow at 37 °C and positive catalase reaction, the observed phenotype was almost indistinguishable from those of A. acidipropionici and A. jensenii. Based on our findings, we conclude that the organism represents a novel member of the genus Acidipropionibacterium, for which we propose the name Acidipropionibacteriumvirtanenii sp. nov. The type strain is JS278T (=VTT E-113202T=DSM 106790T).

Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats.

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor.

Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein.

This note presents the (1)H, (13)C, and (15)N resonances assignment of the 22 kDa, dimeric, C-repressor protein from the P2 bacteriophage. The C-repressor controls the genetic switch that determines if the temperate P2 phage should exist in the lytic or lysogenic lifemode.

A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and WPhi.

Bacteriophages P2 and WPhi are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and WPhi are compared. An in vivo genetic analysis shows that WPhi C, like P2 C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2 C is unable to bind to one half-site as opposed to WPhi, but both half-sites are required for repression of WPhi Pe. A reduction from three to two helical turns between the centers of the half-sites in WPhi has no significant effect on the capacity to repress Pe. However, the protein-DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis.

Bacteriophages P2, P2 Hy dis and WPhi are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein-DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein-DNA complexes, and only WPhi was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WPhi operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WPhi C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.