New Insights into the Genome Organization of Yeast Double-Stranded RNA LBC Viruses

The yeasts Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) may show a killer phenotype that is encoded in dsRNA M viruses (V-M), which require the helper activity of another dsRNA virus (V-LA or V-LBC) for replication. Recently, two TdV-LBCbarr genomes, which share sequence identity with ScV-LBC counterparts, were characterized by high-throughput sequencing (HTS). They also share some similar characteristics with Sc-LA viruses. This may explain why TdV-LBCbarr has helper capability to maintain M viruses, whereas ScV-LBC does not. We here analyze two stretches with low sequence identity (LIS I and LIS II) that were found in TdV-LBCbarr Gag-Pol proteins when comparing with the homologous regions of ScV-LBC. These stretches may result from successive nucleotide insertions or deletions (indels) that allow compensatory frameshift events required to maintain specific functions of the RNA-polymerase, while modifying other functions such as the ability to bind V-M (+)RNA for packaging. The presence of an additional frameshifting site in LIS I may ensure the synthesis of a certain amount of RNA-polymerase until the new compensatory indel appears. Additional 5′- and 3′-extra sequences were found beyond V-LBC canonical genomes. Most extra sequences showed high identity to some stretches of the canonical genomes and can form stem-loop structures. Further, the 3′-extra sequence of two ScV-LBC genomes contains rRNA stretches. The origin and possible functions of these extra sequences are here discussed.

Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.

Genome Organization of a New Double-Stranded RNA LA Helper Virus From Wine Torulaspora delbrueckii Killer Yeast as Compared With Its Saccharomyces Counterparts

Wine killer yeasts such as killer strains of Torulaspora delbrueckii and Saccharomyces cerevisiae contain helper large-size (4.6 kb) dsRNA viruses (V-LA) required for the stable maintenance and replication of killer medium-size dsRNA viruses (V-M) which bear the genes that encode for the killer toxin. The genome of the new V-LA dsRNA from the T. delbrueckii Kbarr1 killer yeast (TdV-LAbarr1) was characterized by high-throughput sequencing (HTS). The canonical genome of TdV-LAbarr1 shares a high sequence identity and similar genome organization with its Saccharomyces counterparts. It contains all the known conserved motifs predicted to be necessary for virus translation, packaging, and replication. Similarly, the Gag-Pol amino-acid sequence of this virus contains all the features required for cap-snatching and RNA polymerase activity, as well as the expected regional variables previously found in other LA viruses. Sequence comparison showed that two main clusters (99.2–100% and 96.3–98.8% identity) include most LA viruses from Saccharomyces, with TdV-LAbarr1 being the most distant from all these viruses (61.5–62.5% identity). Viral co-evolution and cross transmission between different yeast species are discussed based on this sequence comparison. Additional 5′ and 3′ sequences were found in the TdV-LAbarr1 genome as well as in some newly sequenced V-LA genomes from S. cerevisiae. A stretch involving the 5′ extra sequence of TdV-LAbarr1 is identical to a homologous stretch close to the 5′ end of the canonical sequence of the same virus (self-identity). Our modeling suggests that these stretches can form single-strand stem loops, whose unpaired nucleotides could anneal to create an intramolecular kissing complex. Similar stem loops are also found in the 3′ extra sequence of the same virus as well as in the extra sequences of some LA viruses from S. cerevisiae. A possible origin of these extra sequences as well as their function in obviating ssRNA degradation and allowing RNA transcription and replication are discussed.

RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters

Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαβ mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3′-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3′-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.

Structural Examination of Multilayer CrAlSiN/AlSiN Coatings Deposited by Cathodic Arc

In this research, the possibility of applying multilayer multielement super hard coatings by Cathodic Arc is investigated. More precisely the structure of the coating consisting of quaternary CrAlSiN and ternary AlSiN layers is examined by electron microscopy, X-ray diffraction and X-ray photoelectron microscopy analytical methods. The as-deposited samples were found to have distinguishable layers. The CrAlSiN layer is characterized by an extra sequence of repeated nanolayers. The AlSiN layer consisted of nanosized grains having a preferential orientation. Finally the surface layer was found to contain a solid solution of CrxAl1-xN, while Si3N4was identified only by XPS most probably due to its amorphous structure.