bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis
Authors: Laun Peter , Pichova Alena , Madeo Frank , Fuchs Jörg , Ellinger Adolf , Kohlwein Sepp , Dawes Ian , Fröhlich Kai‐Uwe , Breitenbach Michael 
Published: December 2004

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT‐mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro‐apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild‐type yeast mother cells.
A novel group I intron‐encoded endonuclease specific for the anticodon region of tRNAfMet genes
Authors: Bonocora Richard P. , Shub David A. 
Published: December 2004

Open reading frames (ORFs) are frequently inserted into group I self‐splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self‐splicing intron in the tRNAfMet gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intron‐encoded endonucleases or maturases. Here, using an in vitro transcription–translation system, we show that this intronic ORF encodes a double‐strand DNA endonuclease, I‐Ssp6803I. I‐Ssp6803I cleaves each strand of the intronless tRNAfMet gene adjacent to the anticodon triplet leaving 3 bp 3′ extensions and has no activity at intron–exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNAfMet gene. I‐Ssp6803I represents a novel intron‐encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.
Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock
Authors: Weber Michael H. W. , Klein Wolfgang , Müller Lin , Niess Ulf M. , Marahiel Mohamed A. 
Published: December 2004

In our attempt to understand the cold shock response of Bacillus subtilis, we report on the role of the B. subtilis fatty acid desaturase (FA‐D) Des during membrane adaptation to low temperatures and demonstrate its importance during cold shock. A des null mutant was constructed and analysed in comparison with its parental strain. Growth studies and large‐scale comparative fatty acid (FA) analysis revealed a severe cold‐sensitive phenotype of the des deletion mutant during the absence of isoleucine and showed that four unsaturated fatty acid (UFA) species differing in length, branching pattern and position of the double bond are synthesized in B. subtilis JH642 but not in the des null mutant. Apart from the lack of UFA synthesis, the FA‐D deletion strain showed a dramatically altered saturated fatty acid (SFA) profile at the onset of the stationary growth phase in the presence of exogenous isoleucine sources. Expression of des integrated in trans at the amyE locus of the des deletion strain not only cured the cold‐sensitive phenotype observed for the des mutant but allowed much better growth than in strain JH642 after a shift from 37°C to 15°C. These results show that, during cold shock adaptation, des expression can completely replace the isoleucine‐dependent, long‐term, FA branching adaptation mechanism. We conclude that the crucial aspect in cold adaptation of the cytoplasmic membrane is not its specific molecular composition but rather its physical status in terms of its fluidity.
Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways
Authors: Chen Changbin , Dickman Martin B. 
Published: December 2004

SummaryThe small G‐protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient‐dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct‐Rac1), a member of the Rho family of G proteins. Ct‐Rac1 expression is downregulated by DARas under limiting nutrition. Co‐expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild‐type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac‐mediated MAPK activation is responsible for reversion to the wild‐type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH‐DA fluorescence. Co‐expression with DNRac decreases ROS generation to wild‐type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild‐type phenotype. These findings suggest that Ras‐mediated ROS generation is dependent on a Rac–cPLA2‐linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.
Loss of the mono‐allelic control of the VSG expression sites during the development of Trypanosoma brucei in the bloodstream
Authors: Amiguet‐Vercher Amelia , Pérez‐Morga David , Pays Annette , Poelvoorde Philippe , Van Xong Huang , Tebabi Patricia , Vanhamme Luc , Pays Etienne 
Published: December 2004

SummaryTranscription of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei occurs in a single of multiple polycistronic expression sites (ESs). Analysis of RNA from proliferative long slender (LS) bloodstream forms demonstrated that initiation of transcription occurs in different ESs, but inefficient RNA processing and elongation is linked to RNA polymerase arrest in all except one unit at a time. The pattern of ES transcripts was analysed during the transformation of dividing LS forms into quiescent short stumpy (SS) forms. The results demonstrated that the mono‐allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady‐state ES transcripts, particularly the VSG mRNA, were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run‐on transcription analysis indicated that RNA polymerase was still fully associated with the promoter‐proximal half of the ‘active’ ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the ‘active’ ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ. This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.
Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence
Authors: Cabanes Didier , Dussurget Olivier , Dehoux Pierre , Cossart Pascale 
Published: December 2004

Summary Listeria monocytogenes is an opportunistic food‐borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non‐pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N‐terminal autolysin domain and a C‐terminal cell wall‐anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non‐phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.
IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase
Authors: Ilves Heili , Hõrak Rita , Teras Riho , Kivisaar Maia 
Published: December 2004

SummaryTranspositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase‐specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445–5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn4652 as well. Here, we demonstrate that transposition of Tn4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn4652 occurs in a P. putida ihfA‐defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild‐type strain. This indicates that the amount of IHF is a bottleneck in Tn4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase.
Molecular architecture of the Cryptococcus neoformans capsule
Authors: Gates Marcellene A. , Thorkildson Peter , Kozel Thomas R. 
Published: December 2004

SummaryMany microbes are surrounded by phagocytosis‐inhibiting capsules. We took advantage of the large size of the polysaccharide capsule of the pathogenic yeast Cryptococcus neoformans to examine capsular architecture and the relationship between molecular architecture and the interaction of the capsule with potentially opsonic serum proteins. Our experimental design used complementary approaches in which (i) assessment of permeability to macromolecules of different Stokes radii; (ii) determination of the binding of Fab fragments of anticapsular antibodies as a measure of matrix density; (iii) capsular deconstruction by treatment with dimethyl sulphoxide; and (iv) evaluation of capsule plasticity, were used to probe the molecular structure of the capsule. The results showed that the capsule is a matrix with a variable porosity that increases with distance from the cell wall. A high density of the matrix at the capsule interior prevents penetration of large macromolecules to sites near the cell wall. In contrast, the capsular edge that is the interface with phagocytes presents capsular polysaccharide in a very low density that exhibits considerable plasticity and permeability to macromolecules. Notably, the capsule of yeast cells harvested from infected tissue showed a greater matrix density than yeast cells grown in vitro under capsule induction conditions.
A basic‐region helix–loop–helix protein‐encoding gene (devR) involved in the development of Aspergillus nidulans
Authors: Tüncher André , Reinke Hans , Martic Goran , Caruso Maria Louise , Brakhage Axel A. 
Published: December 2004

SummaryBasic‐region helix–loop–helix (bHLH) proteins form an interesting class of eukaryotic transcription factors often involved in developmental processes. Here, a so far unknown bHLH protein‐encoding gene of the filamentous ascomycete Aspergillus nidulans was isolated and designated devR for regulator of development. Deletion of devR revealed that the gene is non‐essential for vegetative growth. However, the deletion mutant produced wrinkled colonies, a yellow pigment and did not form conidia on minimal agar plates. Conidiophore development was initiated normally, and colonies produced conidiophores with metulae and phialides. However, the phialides continued to grow filamentously and produced a second conidiophore with a vesicle at its end. The addition of KCl (0.6 M) to the medium suppressed the knock‐out phenotype. The ΔdevR phenotype resembled that of a mutation in the tcsA gene encoding a histidine kinase domain and a response regulator domain. Here, we generated a tcsA deletion mutant. In a ΔtcsA strain, a DevR–Egfp protein fusion was detected in the cytoplasm, whereas in the wild type, the protein fusion was exclusively located in the nuclei, indicating that TcsA is required for nuclear localization of DevR. devR mRNA steady‐state levels were similar in sporulating and vegetatively growing mycelia, and independent of a functional brlA gene. Moreover, under all conditions tested, self‐crossing of the ΔdevR mutant strain was never observed. Taken together, devR encodes a bHLH regulatory protein that is part of the tcsA signal transduction network and required for development under standard growth conditions.
Zinc is a key factor in controlling alternation of two types of L31 protein in theBacillus subtilisribosome
Authors: Nanamiya Hideaki , Akanuma Genki , Natori Yousuke , Murayama Rikinori , Kosono Saori , Kudo Toshiaki , Kobayashi Kazuo , Ogasawara Naotake , Park Seung‐Moon , Ochi Kozo , Kawamura Fujio 
Published: December 2004

SummaryWe have analysed changes in the composition of ribosomal proteins during cell growth inBacillus subtilis. Ribosome fractions were prepared fromB. subtiliscells at different phases of growth and were separated by radical‐free and highly reducing (RFHR) two‐dimensional polyacrylamide gel electrophoresis. We identified 50 ribosomal proteins, including two paralogues of L31 protein (RpmE and YtiA). Although the ribosome fraction extracted from exponentially growing cells contained RpmE protein, this protein disappeared during the stationary phase. In contrast, YtiA was detected in the ribosome fraction extracted after the end of exponential growth. Expression of theytiAgene encoding YtiA was found to be negatively controlled by Zur, a zinc‐specific transcriptional repressor that controls zinc transport operons. Analysis by inductively coupled plasma mass spectrometry (ICP‐MS) indicated that RpmE contains one zinc ion per molecule of protein. In addition, mutagenesis of therpmEgene encoding RpmE revealed that Cys‐36 and Cys‐39, located within a CxxC motif, are required not only for binding zinc but also for the accumulation of RpmE in the cell. Taken together, these results indicate that zinc plays an essential role in the alternation between two types of L31 protein in the ribosome ofB. subtilis.
Has1p, a member of the DEAD‐box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae†
Authors: Emery Bertrand , De La Cruz Jesús , Rocak Sanda , Deloche Olivier , Linder Patrick 
Published: December 2004

SummaryThe Has1 protein, a member of the DEAD‐box family of ATP‐dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre‐60S ribosomal complexes. Here, we show that Has1p is an essential trans‐acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature‐sensitive has1‐1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre‐rRNA processing by pulse‐chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre‐rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre‐rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high‐molecular‐weight complex sedimenting at positions equivalent to 60S and pre‐60S ribosomal particles. A mutation in the ATP‐binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.
Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes
Authors: Wang Qingfeng , Frye Jonathan G. , McClelland Michael , Harshey Rasika M. 
Published: December 2004

SummarySwarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide ‘slime’. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one‐third of the functional genome. The large number of genes showing surface‐specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming‐specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface‐dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.
clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σH
Authors: Engels Sabine , Schweitzer Jens‐Eric , Ludwig Carsten , Bott Michael , Schaffer Steffen 
Published: December 2004

SummaryThe ATP‐dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes. In Corynebacterium glutamicum, the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit. We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC. Deletion of clgR in the ΔclpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes. ClgR activity itself is probably controlled via ClpC‐dependent regulation of its stability, as ClgR is only present in ΔclpC and not in wild‐type cells, whereas the levels of clgR mRNA are comparable in both strains. clpC, clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR. Identification of the heat‐responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for σECF‐dependent promoters. The ECF sigma factor σH could be identified as being required for transcriptional activation of clpC, clpP1P2 and clgR in response to severe heat stress. A second heat‐responsive but σH‐independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR. Taken together, these results show that clpC and clpP1P2 expression in C. glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli. Both the ClgR‐ and σH‐dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes.
Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus‐mediated perturbation of its phosphorylation in response to hypertonic stress
Authors: Park Seung‐Moon , Choi Eun‐Sil , Kim Myoung‐Ju , Cha Byeong‐Jin , Yang Moon‐Sik , Kim Dae‐Hyuk 
Published: December 2004

SummaryWe examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus. Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1‐homologue from Magnaporthe grisea. A growth defect was observed in the cpmk1‐null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily. Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1‐EP713. Moreover, the virus‐infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus‐free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress. Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence‐associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression. However, the cpmk1‐null mutant exhibited an increased accumulation of pheromone gene transcripts. Virulence assays of the cpmk1‐null mutant revealed reduced canker area, but not as severe as that of UEP1. These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development.