Bacteriophage-resistance mechanisms examined by transfection of Lactococcus lactis subsp. lactis 4513-5 mediated by high voltage electroporation

Summary Phage adsorption tests and transfection by electroporation were carried out to decide whether phage-resistance in Lactococcus lactis subsp. lactis strain 4513-5 is based on intracellular or extracellular mechanisms. Using high voltage (12.5 kV/cm) electroporation, untreated phage DNA was introduced into phage-sensitive and phage-resistant cells. Since phages showed low adsorption frequencies on resistant bacteria, resistance is localized in the cell wall preventing phage DNA from entering the cell. This is the only mechanism responsible for the resistance of L. lactis subsp. lactis 4513-5 against its homologous phage P4513-K12 and non-homologous phages P05M-13 and P05M-47, but not against phage P530-7 and phage P530-12. In the case of the latter two phage strains, intracellular resistance mechanisms are involved and discussed.     

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.     

Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity

A three-dimensional model of yeast alcohol dehydrogenase, based on the homologous horse liver enzyme, was used to compare the substrate binding pockets of the three isozymes (I, II, and III) from Saccharomyces cerevisiae and the enzyme from Schizosaccharomyces pombe. Isozyme I and the S. pombe enzyme have methionine at position 294 (numbered as in the liver enzyme, corresponding to 270 in yeast), whereas isozymes II and III have leucine. Otherwise the active sites of the S. cerevisiae enzymes are the same. All four wild-type enzymes were produced from the cloned genes. In addition, oligonucleotide-directed mutagenesis was used to change Met-294 in alcohol dehydrogenase I to leucine. The mechanisms for all five enzymes were predominantly ordered with ethanol (but partially random with butanol) at pH 7.3 and 30 degrees C. The wild-type alcohol dehydrogenases and the leucine mutant had similar kinetic constants, except that isozyme II had 10-20-fold smaller Michaelis and inhibition constants for ethanol. Thus, residue 294 is not responsible for this difference. Apparently, substitutions outside of the substrate binding pocket indirectly affect the interactions of the alcohol dehydrogenases with ethanol. Nevertheless, the substitution of methionine with leucine in the substrate binding site of alcohol dehydrogenase I produced a 7-10-fold increase in reactivity (V/Km) with butanol, pentanol, and hexanol. The higher activity is due to tighter binding of the longer chain alcohols and to more rapid hydrogen transfer.     

Genome relationship betweenPsathyrostachys huashanica andP. fragilis (Poaceae)

Hybrids between the Chinese endemic speciesPsathyrostachys huashanica Keng and the SW. Asian speciesP. fragilis (Boiss.)Nevski (all 2n = 14) developed normally but were completely sterile. Meiotic analyses revealed a high chiasma frequency indicating that the two species as well asP. juncea (Fisch.)Nevski share the same basic genome (called N). The hybrid nature of the plants was established through karyotype analysis and Giemsa C-banding.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.