Industrially Applicable De Novo Lager Yeast Hybrids with a Unique Genomic Architecture: Creation and Characterization.

Lager beer is produced by Saccharomyces pastorianus, which is a natural allopolyploid hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus Lager strains are classified into two major groups based largely on genomic composition: group I and group II. Group I strains are allotriploid, whereas group II strains are allotetraploid. A lack of phenotypic diversity in commercial lager strains has led to substantial interest in the reconstitution of de novo allotetraploid lager strains by hybridization of S. cerevisiae and S. eubayanus strains. Such strategies rely on the hybridization of wild S. eubayanus isolates, which carry unacceptable traits for commercial lager beer such as phenolic off flavors and incomplete utilization of carbohydrates. Using an alternative breeding strategy, we have created de novo lager hybrids containing the domesticated S. eubayanus subgenome from an industrial S. pastorianus strain by hybridizing diploid meiotic segregants of this strain to a variety of S. cerevisiae ale strains. Five de novo hybrids were isolated which had fermentation characteristics similar to those of prototypical commercial lager strains but with unique phenotypic variation due to the contributions of the S. cerevisiae parents. Genomic analysis of these de novo lager hybrids identified novel allotetraploid genomes carrying three copies of the S. cerevisiae genome and one copy of the S. eubayanus genome. Most importantly, these hybrids do not possess the negative traits which result from breeding wild S. eubayanus The de novo lager strains produced using industrial S. pastorianus in this study are immediately suitable for industrial lager beer production.IMPORTANCE All lager beer is produced using two related lager yeast types: group I and group II, which are highly similar, resulting in a lack of strain diversity for lager beer production. To date, approaches for generating new lager yeasts have generated strains possessing undesirable brewing characteristics which render them commercially inviable. We have used an alternative approach that circumvents this issue and created new lager strains that are directly suitable for lager beer production. These novel lager strains also possess a unique genomic architecture, which may lead to a better understanding of industrial yeast hybrids. We propose that strains created using our approach be classified as a third group of lager strains (group III). We anticipate that these novel lager strains will be of great industrial relevance and that this technique will be applicable to the creation of additional novel lager strains that will help broaden the diversity in commercial lager beer strains.

Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 µM, K(m)(actin) = 24 µM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 µM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.

Photox, a novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens.

Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP-ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46-kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (k(cat), 1680 +/- 75 min(-1)) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha-skeletal actin and nonmuscle beta- and gamma-actin at Arg(177), inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family.

Newly discovered and characterized antivirulence compounds inhibit bacterial mono-ADP-ribosyltransferase toxins.

The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins from Pseudomonas aeruginosa and Vibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.

Cholera- and anthrax-like toxins are among several new ADP-ribosyltransferases.

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences--including a primary sequence pattern--to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data--and we need high-throughput validation--our approach provides insight into the newest toxin ADP-ribosyltransferases.

Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins.

The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.