scholarly journals Roles of Phosphatidylethanolamine and of Its Several Biosynthetic Pathways inSaccharomyces cerevisiae

2001 ◽  
Vol 12 (4) ◽  
pp. 997-1007 ◽  
Author(s):  
Ruth Birner ◽  
Maria Bürgermeister ◽  
Roger Schneiter ◽  
Günther Daum
Keyword(s):  
Biochemical Analysis ◽  
Membrane Vesicle ◽  
Carbon Sources ◽  
Carboxypeptidase Y ◽  
Deletion Mutants ◽  
Wild Type ◽  
Inner Mitochondrial Membrane ◽  
Minimal Level ◽  
Delayed Maturation

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. Inpsd1Δ strains or cho1Δ strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

scholarly journals Sinorhizobium meliloti Mutants Lacking Phosphotransferase System Enzyme HPr or EIIA Are Altered in Diverse Processes, Including Carbon Metabolism, Cobalt Requirements, and Succinoglycan Production

2008 ◽  
Vol 190 (8) ◽  
pp. 2947-2956 ◽  
Author(s):  
Catalina Arango Pinedo ◽  
Ryan M. Bringhurst ◽  
Daniel J. Gage
Keyword(s):  
Carbon Metabolism ◽  
Sinorhizobium Meliloti ◽  
Carbon Sources ◽  
Deletion Mutants ◽  
Symbiotic Relationship ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Carbon Utilization ◽  
Carbon Regulation ◽  
Extreme Sensitivity

ABSTRACT Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIAMan-type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of α- and β-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecular-weight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIAMan) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.

scholarly journals Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks

2001 ◽  
Vol 75 (8) ◽  
pp. 3811-3818 ◽  
Author(s):  
Mengji Lu ◽  
Gero Hilken ◽  
Dongliang Yang ◽  
Thekla Kemper ◽  
Michael Roggendorf
Keyword(s):  
Hepatitis Virus ◽  
Viral Population ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus ◽  
Deletion Mutants ◽  
Wild Type ◽  
Lymphoproliferative Response ◽  
Hepatocyte Cultures ◽  
Liver Tissues

ABSTRACT Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

scholarly journals Catalase protects against non-enzymatic decarboxylations during photorespiration

2021 ◽  
Author(s):  
Han Bao ◽  
Matt Morency ◽  
Winda Rianti ◽  
Sompop Saeheng ◽  
Sanja Roje ◽  
...  
Keyword(s):  
Gas Exchange ◽  
Biochemical Analysis ◽  
Co2 Assimilation ◽  
Wild Type ◽  
Co2 Compensation Point ◽  
Metabolic Products ◽  
Alternative Hypotheses ◽  
Co2 Release ◽  
Enzymatic Decarboxylation

Photorespiration recovers carbon that would be otherwise lost following the oxygenation reaction of rubisco and production of glycolate. Photorespiration is essential in plants and recycles glycolate into usable metabolic products through reactions spanning the chloroplast, mitochondrion, and peroxisome. Catalase in peroxisomes plays an important role in this process by disproportionating H2O2 resulting from glycolate oxidation into O2 and water. We hypothesize that catalase in the peroxisome also protects against non-enzymatic decarboxylations between hydrogen peroxide and photorespiratory intermediates (glyoxylate and/or hydroxypyruvate). We test this hypothesis by detailed gas exchange and biochemical analysis of Arabidopsis thaliana mutants lacking peroxisomal catalase. Our results strongly support this hypothesis, with catalase mutants showing gas exchange evidence for an increased stoichiometry of CO2 release from photorespiration, specifically an increase in the CO2 compensation point, a photorespiratory-dependent decrease in the quantum efficiency of CO2 assimilation, increase in the 12CO2 released in a 13CO2 background and an increase in the post-illumination CO2 burst. Further metabolic evidence suggests this excess CO2 release occurred via the non-enzymatic decarboxylation of hydroxypyruvate. Specifically, the catalase mutant showed an accumulation of photorespiratory intermediates during a transient increase in rubisco oxygenation consistent with this hypothesis. Additionally, end products of alternative hypotheses explaining this excess release were similar between wild type and catalase mutants. Furthermore, the calculated rate of hydroxypyruvate decarboxylation in catalase mutant is much higher than that of glyoxylate decarboxylation. This work provides evidence that these non-enzymatic decarboxylation reactions, predominately hydroxypyruvate decarboxylation, can occur in vivo when photorespiratory metabolism is genetically disrupted.

scholarly journals Recombinant Human Metapneumovirus Lacking the Small Hydrophobic SH and/or Attachment G Glycoprotein: Deletion of G Yields a Promising Vaccine Candidate

2004 ◽  
Vol 78 (23) ◽  
pp. 12877-12887 ◽  
Author(s):  
Stéphane Biacchesi ◽  
Mario H. Skiadopoulos ◽  
Lijuan Yang ◽  
Elaine W. Lamirande ◽  
Kim C. Tran ◽  
...  
Keyword(s):  
Cell Culture ◽  
Respiratory Tract ◽  
Human Metapneumovirus ◽  
Reverse Genetic System ◽  
Deletion Mutants ◽  
Upper Respiratory Tract ◽  
Respiratory Tract Disease ◽  
Wild Type ◽  
Tract Disease

ABSTRACT Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The ΔSH, ΔG, and ΔSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the ΔG and ΔSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the ΔG and ΔSH/G viruses represent promising vaccine candidates. In contrast, ΔSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.

scholarly journals Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 1115-1124 ◽  
Author(s):  
Margaret A. Kenna ◽  
Carrie Baker Brachmann ◽  
Scott E. Devine ◽  
Jef D. Boeke
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Reverse Transcriptase Activity ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Localization Signal

ABSTRACT Retrotransposon Ty1 faces a formidable cell barrier during transposition—the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.

scholarly journals Alteration of a Novel Dispensable Mitochondrial Ribosomal Small-Subunit Protein, Rsm28p, Allows Translation of Defective COX2 mRNAs

2005 ◽  
Vol 4 (2) ◽  
pp. 337-345 ◽  
Author(s):  
Elizabeth H. Williams ◽  
Nada Bsat ◽  
Nathalie Bonnefoy ◽  
Christine A. Butler ◽  
Thomas D. Fox
Keyword(s):  
Mitochondrial Gene ◽  
Carbon Sources ◽  
Small Subunit ◽  
Wild Type ◽  
Inner Mitochondrial Membrane ◽  
Positive Role ◽  
Mitochondrial Ribosomes ◽  
The Matrix ◽  
Triton X ◽  
Type Strains

ABSTRACT Mutations affecting the RNA sequence of the first 10 codons of the Saccharomyces cerevisiae mitochondrial gene COX2 strongly reduce translation of the mRNA, which encodes the precursor of cytochrome c oxidase subunit II. A dominant chromosomal mutation that suppresses these defects is an internal in-frame deletion of 67 codons from the gene YDR494w. Wild-type YDR494w encodes a 361-residue polypeptide with no similarity to proteins of known function. The epitope-tagged product of this gene, now named RSM28, is both peripherally associated with the inner surface of the inner mitochondrial membrane and soluble in the matrix. Epitope-tagged Rsm28p from Triton X-100-solubilized mitochondria sedimented with the small subunit of mitochondrial ribosomes in a sucrose gradient containing 500 mM NH4Cl. Complete deletion of RSM28 caused only a modest decrease in growth on nonfermentable carbon sources in otherwise wild-type strains and enhanced the respiratory defect of the suppressible cox2 mutations. The rsm28 null mutation also reduced translation of an ARG8 m reporter sequence inserted at the COX1, COX2, and COX3 mitochondrial loci. We tested the ability of RSM28-1 to suppress a variety of cox2 and cox3 mutations and found that initiation codon mutations in both genes were suppressed. We conclude that Rsm28p is a dispensable small-subunit mitochondrial ribosomal protein previously undetected in systematic investigations of these ribosomes, with a positive role in translation of several mitochondrial mRNAs.

scholarly journals Selective Removal of Aberrant Extender Units by a Type II Thioesterase for Efficient FR-008/Candicidin Biosynthesis in Streptomyces sp. Strain FR-008

2008 ◽  
Vol 74 (23) ◽  
pp. 7235-7242 ◽  
Author(s):  
Yongjun Zhou ◽  
Qingqing Meng ◽  
Delin You ◽  
Jialiang Li ◽  
Shi Chen ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Biochemical Analysis ◽  
Wild Type Strain ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Acyl Carrier Proteins ◽  
Peptide Synthetase ◽  
In The Wild

ABSTRACT Gene fscTE, encoding a putative type II thioesterase (TEII), was associated with the FR-008/candicidin gene cluster. Deletion of fscTE reduced approximately 90% of the FR-008/candicidin production, while the production level was well restored when fscTE was added back to the mutant in trans. FscTE was unable to compensate for the release of the maturely elongated polyketide as site-directed inactivation of the type I thioesterase (TEI) totally abolished FR-008/candicidin production. Direct biochemical analysis of FscTE in parallel with its homologue TylO from the tylosin biosynthetic pathway demonstrated their remarkable preferences for acyl-thioesters (i.e., propionyl-S-N-acetylcysteamine [SNAC] over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC) and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to acyl carrier proteins. Overexpression of FscTE under the strong constitutive ermE*p promoter in the wild-type strain did not suppress FR-008/candicidin formation, which confirmed its substrate specificity in vivo. Furthermore, successful complementation of the fscTE mutant was obtained with fscTE and tylO, whereas no complementation was detected with nonribosomal peptide synthetase (NRPS) TEII tycF and srfAD, reflecting substrate specificities of TEIIs distinctive from those of either polyketide synthases or NRPSs.

scholarly journals Identification and Characterization of theNeisseria gonorrhoeaeMscS-Like Mechanosensitive Channel

2018 ◽  
Vol 86 (6) ◽  
Author(s):  
Zhemin Wang ◽  
Xiaomin Wang ◽  
Ping Lu ◽  
Chunshan Ni ◽  
Yuezhou Li ◽  
...  
Keyword(s):  
Turgor Pressure ◽  
Bacterial Pathogen ◽  
Mechanosensitive Channel ◽  
Infection Model ◽  
Deletion Mutants ◽  
Mechanosensitive Channels ◽  
Wild Type ◽  
Content Type ◽  
Type Strains

ABSTRACTMechanosensitive channels are ubiquitous in bacteria and provide an essential mechanism to survive sudden exposure to a hypo-osmotic environment by the sensing and release of increased turgor pressure. No mechanosensitive channels have thus far been identified and characterized for the human-specific bacterial pathogenNeisseria gonorrhoeae. In this study, we identified and characterized theN. gonorrhoeaeMscS-like mechanosensitive channel (Ng-MscS). Electrophysiological analyses by the patch clamp method showed that Ng-MscS is stretch activated and contains pressure-dependent gating properties. Further mutagenesis studies of critical residues forming the hydrophobic vapor lock showed that gain-of-function mutations in Ng-MscS inhibited bacterial growth. Subsequent analysis of the function of Ng-MscS inN. gonorrhoeaeby osmotic down-shock assays revealed that the survival of Ng-mscSdeletion mutants was significantly reduced compared with that of wild-type strains, while down-shock survival was restored upon the ectopic complementation ofmscS. Finally, to investigate whether Ng-MscS is important forN. gonorrhoeaeduring infections, competition assays were performed by using a murine vaginal tract infection model. Ng-mscSdeletion mutants were outcompeted byN. gonorrhoeaewild-type strains for colonization and survival in this infection model, highlighting that Ng-MscS contributes toin vivocolonization and survival. Therefore, Ng-MscS might be a promising target for the future development of novel antimicrobials.

scholarly journals 5′ termini of polyoma virus early region transcripts synthesized in vivo by wild-type virus and viable deletion mutants

1982 ◽  
Vol 159 (2) ◽  
pp. 189-224 ◽  
Author(s):  
Robert Kamen ◽  
Parmjit Jat ◽  
Richard Treisman ◽  
Jennifer Favaloro ◽  
William R. Folk
Keyword(s):  
Polyoma Virus ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Type Virus ◽  
Wild Type ◽  
Early Region

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

2008 ◽  
Vol 89 (5) ◽  
pp. 1220-1224 ◽  
Author(s):  
Mark P. Zwart ◽  
Eloy Erro ◽  
Monique M. van Oers ◽  
J. Arjan G. M. de Visser ◽  
Just M. Vlak
Keyword(s):  
Cell Culture ◽  
Purifying Selection ◽  
Serial Passage ◽  
Autographa Californica ◽  
Homologous Region ◽  
Deletion Mutants ◽  
Wild Type ◽  
Insect Larvae ◽  
Selection For

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective condition.

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