Post-transcriptional regulation of glutamate metabolism of Pichia pastoris and development of a glutamate-inducible yeast expression system

Pichia pastoris harbours a unique glutamate utilization pathway in which glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the conversion of glutamate to α-ketoglutarate, oxaloacetate, and phosphoenolpyruvate respectively in the cytosol. GDH2 and PEPCK are glutamate-inducible enzymes and their synthesis is regulated post-transcriptionally by Rtg1p, a cytosolic basic helix-loop-helix protein via Rtg1p response elements located downstream of TATA box of GDH2 and PEPCK promoters. Glutamate-inducible synthesis of PEPCK is abrogated in Δgdh2 and Δaat2. α-ketoglutarate induces PEPCK synthesis in Δgdh2 but not Δaat2. We propose that oxaloacetate derived from glutamate is the inducer of PEPCK synthesis. Enzymes of glutamate utilization pathway are synthesized during carbon starvation and they enable P. pastoris to overcome nutritional stress. Finally, green fluorescent protein can be synthesized efficiently from GDH2 and PEPCK promoters using food-grade monosodium glutamate as inducer indicating that the post-transcriptional regulatory circuit described here can be exploited for the development of glutamate-inducible P. pastoris expression system.

Ebulin l Is Internalized in Cells by Both Clathrin-Dependent and -Independent Mechanisms and Does Not Require Clathrin or Dynamin for Intoxication

Ebulin l is an A-B toxin, and despite the presence of a B chain, this toxin displays much less toxicity to cells than the potent A-B toxin ricin. Here, we studied the binding, mechanisms of endocytosis, and intracellular pathway followed by ebulin l and compared it with ricin. COS-1 cells and HeLa cells with inducible synthesis of a mutant dynamin (K44A) were used in this study. The transport of these toxins was measured using radioactively or fluorescently labeled toxins. The data show that ebulin l binds to cells to a lesser extent than ricin. Moreover, the expression of mutant dynamin does not affect the endocytosis, degradation, or toxicity of ebulin l. However, the inhibition of clathrin-coated pit formation by acidification of the cytosol reduced ebulin l endocytosis but not toxicity. Remarkably, unlike ricin, ebulin l is not transported through the Golgi apparatus to intoxicate the cells and ebulin l induces apoptosis as the predominant cell death mechanism. Therefore, after binding to cells, ebulin l is taken up by clathrin-dependent and -independent endocytosis into the endosomal/lysosomal system, but there is no apparent role for clathrin and dynamin in productive intracellular routing leading to intoxication.

Influence of temperature processing on the constitutive and inducible resistance of rye seedlings to chloride salinization

We studied the growth rate of rye seedlings, as well as the dynamics of the content of soluble proteins and proline in the shoots during their adaptation to sharp (300 mM NaCl once, exposure time 9 days) and gradual (100 mM NaCl, then 100 mM NaCl after 2 days to the final concentration of 400 mM) salinity with sodium chloride in the presence or absence of thermal hardening (+40°C, 3 h). The established dy-namics of the content of proline and soluble proteins in the shoots suggests that the formation of re-sistance to salinity is determined by the high constitutive level of proline, as well as the stress-inducible synthesis of proline and water-soluble proteins. Thermal pretreatment of the seedlings stimulated their constitutive stability to a greater extent. The detected metabolic changes are obviously related to one of the possible mechanisms of the protective effect of thermal hardening on subsequent salinization.

Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium

ABSTRACTThe alphaproteobacteriumHyphomonas neptuniumproliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies twoH. neptuniumpromoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions inH. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species.

Cross Talk between PKC and CREB in the Induction of COX-2 by PGF2α in Human Amnion Fibroblasts

Compelling evidence indicates a crucial role of prostaglandin F2α (PGF2α) in parturition. Both the maternal and fetal sides of the fetal membranes synthesize PGF2α, which exerts effects via the prostaglandin F2α receptor (FP) that is coupled to the activation of protein kinase C (PKC). Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step of the inducible synthesis of prostaglandin. Although activation of PKC is known to induce COX-2 expression, it is not clear whether PGF2α can induce COX-2 via FP receptor-coupled PKC activation. COX-2 promoter carries a cAMP-response element (CRE) and phosphorylation of CRE binding protein 1 (CREB1) is associated with COX-2 expression in human amnion fibroblasts. We demonstrated that human amnion fibroblasts produced PGF2α and expressed FP receptor. PGF2α increased COX-2 expression and CREB1 phosphorylation, which could be blocked by either the FP receptor antagonist AL8810 or PKC inhibitor Ro31-7549. The PKC activator, phorbol-12-myristate-13-acetate (PMA), could mimic the induction of COX-2 and CREB1 phosphorylation. The induction of COX-2 by PGF2α and PMA could be attenuated by the small interfering RNA-mediated knockdown of CREB1 expression or overexpressing dominant-negative CREB1. A chromatin immunoprecipitation assay showed that the binding of CREB1 to the COX-2 promoter was increased by PGF2α and PMA in amnion fibroblasts. In conclusion, we provide evidence that PGF2α induces COX-2 expression via the FP receptor and phosphorylates CREB1 by PKC, thus increasing CREB1 binding to the COX-2 promoter and the expression of COX-2 in human amnion fibroblasts. This feed-forward loop may be crucial for the production of prostaglandins in the fetal membranes prior to the onset of labor.

vanM, a New Glycopeptide Resistance Gene Cluster Found in Enterococcus faecium

ABSTRACT Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, 96 μg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-d-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in d-Ala-d-Lac.