Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their Nterminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a ␤1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2⌬. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.The two major components of the cell wall of the yeast Saccharomyces cerevisiae are glucan, which constitutes the inner layer of the cell wall, and mannoproteins, which are embedded in and cover this glucan layer. Chitin is a minor component of the cell wall (13, 18). The mannoproteins can be divided into two groups, the sodium dodecyl sulfate (SDS)-extractable mannoproteins and the glucanase-extractable mannoproteins, which are solubilized by glucanase digestion of the glucan layer. Several glucanase-extractable mannoproteins have been identified. These proteins have two characteristics in common: their C-terminal regions are rich in serine and threonine, and they all contain putative glycosylphosphatidylinositol (GPI) attachment signals. Two of these proteins, ␣-agglutinin (22) and the core subunit of a-agglutinin (33), are involved in mating. The third, which is involved in flocculation, is the product of the FLOI gene (42). Because of the high serine and threonine content of their C-terminal regions, these proteins are probably heavily O glycosylated, which could give the protein a rod-like structure (15). The presence of a GPI anchor has been demonstrated only for the intracellular precursor form of ␣-agglutinin (47). The glucanase-extractable mannoproteins are proposed to be covalently linked to glucan (29,38,45). Several groups have investigated which part of the protein is responsible for anchoring the protein...

Next Generation Sequencing (NGS) combined with powerful bioinformatic approaches are revolutionising food microbiology. Whole genome sequencing (WGS) of single isolates allows the most detailed comparison possible hitherto of individual strains. The two principle approaches for strain discrimination, single nucleotide polymorphism (SNP) analysis and genomic multi-locus sequence typing (MLST) are showing concordant results for phylogenetic clustering and are complementary to each other. Metabarcoding and metagenomics, applied to total DNA isolated from either food materials or the production environment, allows the identification of complete microbial populations. Metagenomics identifies the entire gene content and when coupled to transcriptomics or proteomics, allows the identification of functional capacity and biochemical activity of microbial populations. The focus of this review is on the recent use and future potential of NGS in food microbiology and on current challenges. Guidance is provided for new users, such as public health departments and the food industry, on the implementation of NGS and how to critically interpret results and place them in a broader context. The review aims to promote the broader application of NGS technologies within the food industry as well as highlight knowledge gaps and novel applications of NGS with the aim of driving future research and increasing food safety outputs from its wider use.

The 2011 East Japan earthquake generated a massive tsunami that launched an extraordinary transoceanic biological rafting event with no known historical precedent. We document 289 living Japanese coastal marine species from 16 phyla transported over 6 years on objects that traveled thousands of kilometers across the Pacific Ocean to the shores of North America and Hawai'i. Most of this dispersal occurred on nonbiodegradable objects, resulting in the longest documented transoceanic survival and dispersal of coastal species by rafting. Expanding shoreline infrastructure has increased global sources of plastic materials available for biotic colonization and also interacts with climate change-induced storms of increasing severity to eject debris into the oceans. In turn, increased ocean rafting may intensify species invasions.

Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbJ2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was completed very near to initiation of DNA synthesis. However, DNA synthesis in the mutant was only delayed at 37°C, and the cells blocked in nuclear division. Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division. DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division.In the budding yeast Saccharomyces cerevisiae, the events in the cell cycle from the beginning, START, to the initiation of DNA synthesis were originally defined by three cell cycle mutants. In order of function, these are cdc28, cdc4, and cdc7 (for reviews, see references 25, 30, and 36). Recent molecular analysis of these genes suggests that progress through G1 up to and including initiation of DNA synthesis may be regulated by protein modification, specifically phosphorylation (22, 23). The CDC28 gene product is a protein kinase (31), and CDC4 has homology with the oncogene ets (29). In addition, CDC7, which functions very close to the beginning of DNA synthesis and is probably required directly in initiation (10), also encodes a protein kinase (1,27). At present, nothing is known of the substrates of CDC7 or its actual role in initiation, but it may be significant that at least one of the components of the yeast replication complex is phosphorylated and a protein kinase activity that is temperature sensitive in cdc7 mutant strains is also found in this complex (14).There are likely to be further essential G1 events, other than those defined by cdc4 and cdc7, connected with Sphase control and initiation of DNA synthesis. With this in mind, we isolated new cell cycle mutants and screened them for defects in DNA synthesis under restrictive conditions (17). Four mutants, dbfl to 4, were identified specifically as being defective in DNA synthesis, either in initiation or in DNA replication itself. DBFI is required for ongoing replication, and the gene has been cloned and sequenced, but its product has no homology with any known proteins in the data banks (manuscript in preparation). The DBF4 gene acts between cdc4 and cdc7 (18); it has been cloned and sequenced (our unpublished result), and Northern (RNA) * Corresponding author. t Present address: Distillers Co...