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Anaerobic ammonium oxidation (anammox) is an ecologically and industrially important process and is performed by a clade of deeply branching Planctomycetes . Anammox bacteria possess an intracytoplasmic membrane-bounded organelle, the anammoxosome. In the present study, the ultrastructures of four different genera of anammox bacteria were compared with transmission electron microscopy and electron tomography. The four anammox genera shared a common cell plan and contained glycogen granules. Differences between the four genera included cell size (from 800 to 1,100 nm in diameter), presence or absence of cytoplasmic particles, and presence or absence of pilus-like appendages. Furthermore, cytochrome c proteins were detected exclusively inside the anammoxosome. This detection provides further support for the hypothesis that this organelle is the locus of anammox catabolism.

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis .

Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama VHH antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion. Eukaryotic cells, such as yeasts, have several quality control systems to ensure that only correctly folded proteins are trans- ported along their secretory pathway (9, 13, 20). These quality control systems are necessary because protein folding in vivo is much more complex than protein folding in vitro. The nascent polypeptide chain emerges from the ribosome and is translo- cated across the endoplasmic reticulum (ER) membrane, thereby being gradually exposed to the lumen of the ER. Since