C. Abrusci

Application of Molecular Techniques to the Elucidation of the Microbial Community Structure of Antique Paintings

This paper uses molecular techniques to describe the microstructure and microbiological communities of sixteenth century artwork and their relationships. The microbiological populations, analysed by denaturing gradient gel electrophoresis (DGGE), were highly influenced by the chemical composition of the pictorial layers detected by energy-dispersive X-ray analysis. DGGE revealed that the diversity of microbial communities was lower in pictorial layers composed of pigments with metals, such as Pb, Cu and Hg, than in those found in pictorial layers without such compounds. The number of cultivable microorganisms, mainly fungi and bacteria, was very low in comparison to those found by DGGE, revealing the presence of both cultivable and as-yet-uncultivated (or not viable) species in the samples analysed. Both fungi and bacteria were present in a non-random spatial distribution. Environmental scanning electron microscopy and fluorescent in situ hybridisation analyses revealed that bacterial populations were usually found in close contact with the surface of the pictorial layers, and fungal populations were located on the bacterial biofilm. This work shows, for the first time, the correlation between the diversity of the microbial populations and the chemical composition of the pictorial layers of an artwork.

Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

PMKT is a channel‐forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2Δ mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT‐affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring‐defective mutants were much less sensitive to PMKT, as were wild‐type protoplasts pretreated with phosphatidylinositol‐specific phospholipase C to remove GPI‐anchored proteins, indicating that the GPI‐anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein‐entrapped liposomes containing a purified GFP–Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein‐free liposomes. Cwp2p and its GPI‐anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.

The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.