http://hzi.openrepository.com:80/hzi HZI captures, stores, indexes, preserves, and distributes digital research material. Wed, 22 May 2013 19:21:27 GMT 2013-05-22T19:21:27Z http://hdl.handle.net/10033/292559 Title: Identification of the N-terminal domain of the influenza virus PA responsible for the suppression of host protein synthesis. Authors: Desmet, Emily A; Bussey, Kendra A; Stone, Raychel; Takimoto, Toru Abstract: Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA's reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis. Fri, 01 Mar 2013 00:00:00 GMT http://hdl.handle.net/10033/292559 2013-03-01T00:00:00Z http://hdl.handle.net/10033/291142 Title: The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis. Authors: Dahl, Jan-Ulrik; Radon, Christin; Bühning, Martin; Nimtz, Manfred; Leichert, Lars I; Denis, Yann; Jourlin-Castelli, Cécile; Iobbi-Nivol, Chantal; Méjean, Vincent; Leimkühler, Silke Abstract: The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes. Fri, 22 Feb 2013 00:00:00 GMT http://hdl.handle.net/10033/291142 2013-02-22T00:00:00Z http://hdl.handle.net/10033/291139 Title: Sphingomonas starnbergensis sp. nov., isolated from a prealpine freshwater lake. Authors: Chen, Hong; Jogler, Mareike; Tindall, Brian J; Klenk, Hans-Peter; Rohde, Manfred; Busse, Hans-Jürgen; Overmann, Jörg Abstract: A novel type of freshwater bacterium was isolated from the prealpine mesotrophic Starnberger See (Bavaria, southern Germany). Cells of strain 382(T) were Gram-negative and rod-shaped, motile and creamy-white. The isolate was strictly aerobic, catalase- and oxidase-positive, and grew at pH values of 6-9 (optimum, pH 7) and temperatures of 10-37 °C (optimum, 28 °C). The genomic G+C content of strain 382(T) was 64.1 mol%. Based on 16S rRNA gene sequence analyses, strain 382(T) belongs to the family Sphingomonadaceae and clusters within the genus Sphingomonas. Sphingomonas histidinilytica UM 2(T) and Sphingomonas wittichii DSM 6014(T) were the closest relatives, as indicated by the highest 16S rRNA gene sequence similarities (97.1 % and 96.8 %, respectively). Sphingomonas paucimobilis DSM 1098(T) (the type species of the genus Sphingomonas) exhibited 95.3 % sequence similarity. This affiliation of strain 382(T) to the genus Sphingomonas is confirmed by the presence of Q-10 as the major respiratory quinone, two sphingoglycolipids, C14 : 0 2-OH as the major 2-hydroxy fatty acid and sym-homospermidine as the major polyamine. The main cellular fatty acids of strain 382(T) were C18 : 1ω7c (39 %), C16 : 1ω7c (21 %), C16 : 0 (10 %) and C14 : 0 2-OH (10 %). Based on the phylogenetic distance from other species of the genus Sphingomonas and its unusually high C16 : 1ω7c content, strain 382(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas starnbergensis is proposed. The type strain is 382(T) ( = DSM 25077(T)  = LMG 26763(T)). Fri, 01 Mar 2013 00:00:00 GMT http://hdl.handle.net/10033/291139 2013-03-01T00:00:00Z http://hdl.handle.net/10033/291116 Title: The human cytomegalovirus UL51 protein is essential for viral genome cleavage-packaging and interacts with the terminase subunits pUL56 and pUL89. Authors: Borst, Eva Maria; Kleine-Albers, Jennifer; Gabaev, Ildar; Babic, Marina; Wagner, Karen; Binz, Anne; Degenhardt, Inga; Kalesse, Markus; Jonjic, Stipan; Bauerfeind, Rudolf; Messerle, Martin Abstract: Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle. Fri, 01 Feb 2013 00:00:00 GMT http://hdl.handle.net/10033/291116 2013-02-01T00:00:00Z