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Deletion Strain (deletion + strain)
Selected AbstractsConformational properties of bacterial DnaK and yeast mitochondrial Hsp70FEBS JOURNAL, Issue 12 2005-helical subdomain, Role of the divergent C-terminal Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK/mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42 °C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal ,-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions. [source] Involvement of thiaminase II encoded by the THI20 gene in thiamin salvage of Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2008Mari Onozuka Abstract The physiological significance of thiaminase II, which catalyzes the hydrolysis of thiamin, has remained elusive for several decades. The C-terminal domains of THI20 family proteins (THI20/21/22) and the whole region of PET18 gene product of Saccharomyces cerevisiae are homologous to bacterial thiaminase II. On the other hand, the N-terminal domains of THI20 and THI21 encode 2-methyl-4-amino-5-hydroxymethylpyrimidine kinase and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase involved in the thiamin synthetic pathway. In this study, it was first indicated that the C-terminal domains of the THI20 family and PET18 are not required for de novo thiamin synthesis in S. cerevisiae, using a quadruple deletion strain expressing the N-terminal domain of THI20. Biochemical analysis using cell-free extracts and recombinant proteins demonstrated that yeast thiaminase II activity is exclusively encoded by THI20. It appeared that Thi20p has an affinity for the pyrimidine moiety of thiamin, and HMP produced by the thiaminase II activity is immediately phosphorylated. Thi20p was found to participate in the formation of thiamin from two synthetic antagonists, pyrithiamin and oxythiamin, by hydrolyzing both antagonists and phosphorylating HMP to give HMP pyrophosphate. Furthermore, 2-methyl-4-amino-5-aminomethylpyrimidine, a presumed naturally occurring thiamin precursor, was effectively converted to HMP by incubation with Thi20p. It is proposed that the thiaminase II activity of Thi20p is involved in the thiamin salvage pathway by catalyzing the hydrolysis of HMP precursors in S. cerevisiae. [source] The transcarboxylase domain of pyruvate carboxylase is essential for assembly of the peroxisomal flavoenzyme alcohol oxidaseFEMS YEAST RESEARCH, Issue 7 2007Paulina Z. Ozimek Abstract Pyruvate carboxylase (Pyc1p) has multiple functions in methylotrophic yeast species. Besides its function as an enzyme, Pyc1p is required for assembly of peroxisomal alcohol oxidase (AO). Hence, Pyc1p-deficient cells share aspartate auxotrophy (Asp,) with a defect in growth on methanol as sole carbon source (Mut,). To identify regions in Hansenula polymorpha Pyc1p that are required for the function of HpPyc1p in AO assembly, a series of random mutations was generated in the HpPYC1 gene by transposon mutagenesis. Upon introduction of 18 mutant genes into the H. polymorpha PYC1 deletion strain (pyc1), four different phenotypes were obtained, namely Asp, Mut,, Asp, Mut+, Asp+ Mut,, and Asp+ Mut+. One mutant showed an Asp+ Mut, phenotype. This mutant produced HpPyc1p containing a pentapeptide insertion in the region that links the conserved N-terminal biotin carboxylation domain (BC) with the central transcarboxylation (TC) domain. Three mutants that were Asp, Mut, contained insertions in the TC domain, suggesting that this domain is important for both functions of Pyc1p. Analysis of a series of constructed C-terminal and N-terminal truncated versions of HpPyc1p showed that the TC domain of Pyc1p, including the region linking this domain to the BC domain, is essential for AO assembly. [source] Gluconeogenesis in Candida albicansFEMS YEAST RESEARCH, Issue 3 2002D. Eschrich Abstract According to different metabolic situations in various stages of Candida albicans pathogenesis the regulation of carbohydrate metabolism was investigated. We report the genetic characterization of all major C. albicans gluconeogenic and glyoxylate cycle genes (fructose-1,6-bisphosphatase, PEP carboxykinase, malate synthase and isocitrate lyase) which were isolated after functional complementation of the corresponding Saccharomyces cerevisiae deletion mutants. Remarkably, the regulation of the heterologously expressed C. albicans gluconeogenic and glyoxylate cycle genes was similar to that of the homologous S. cerevisiae genes. A C. albicans,Cafbp1 deletion strain failed to utilize non-fermentable carbon sources but hyphal growth was not affected. Our results show that regulation of gluconeogenesis in C. albicans is similar to that of S. cerevisiae and that the current knowledge on how gluconeogenesis is regulated will facilitate the physiological understanding of C. albicans. [source] Comparative analysis of changes in gene expression due to RNA melting activities of translation initiation factor IF1 and a cold shock protein of the CspA familyGENES TO CELLS, Issue 11 2009Sangita Phadtare In Escherichia coli, temperature downshift elicits cold shock response, which is characterized by induction of cold shock proteins. CspA, the major cold shock protein of E. coli, helps cells to acclimatize to low temperature by melting the secondary structures in nucleic acids and acting as a transcription antiterminator. CspA and its homologues contain the cold shock domain and belong to the oligomer binding protein family, which also includes S1 domain proteins such as IF1. Structural similarity between IF1 and CspA homologues suggested a functional overlap between these proteins. Indeed IF1 can melt secondary structures in RNA and acts as transcription antiterminator in vivo and in vitro. Here, we show that in spite of having these critical activities, IF1 does not complement cold-sensitivity of a csp quadruple deletion strain. DNA microarray analysis shows that overproduction of IF1 and Csp leads to changes in expression of different sets of genes. Importantly, several genes which were previously shown to require Csp proteins for their expression at low temperature did not respond to IF1. Moreover, in vitro, we show that a transcription terminator responsive to Csp does not respond to IF1. Our results suggest that Csp proteins and IF1 have different sets of target genes as they may be suppressing the function of different types of transcription termination elements in specific genes. [source] Specific and pleiotropic patterns of mRNA regulation by ArcZ, a conserved, Hfq-dependent small RNAMOLECULAR MICROBIOLOGY, Issue 1 2009Kai Papenfort Summary The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome-wide screens in Escherichia coli and Salmonella. Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial ,core' small RNA, yet its function remained unknown. Here, we report that ArcZ acts as a post-transcriptional regulator in Salmonella, repressing the mRNAs of the widely distributed sdaCB (serine uptake) and tpx (oxidative stress) genes, and of STM3216, a horizontally acquired methyl-accepting chemotaxis protein (MCP). Both sdaCB and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the tpx mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an arcZ deletion strain further argued for the existence of a distinct set of Salmonella loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady-state levels of > 16% (> 750) of all Salmonella mRNAs, and rendered the bacteria non-motile. Deep sequencing detected a dramatically changed profile of Hfq-bound sRNAs and mRNAs, suggesting that the unprecedented pleiotropic effects by a single sRNA might in part be caused by altered post-transcriptional regulation. [source] The RNA chaperone Hfq is essential for the virulence of Salmonella typhimuriumMOLECULAR MICROBIOLOGY, Issue 1 2007Alexandra Sittka Summary The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Q, RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression. [source] Ribosomal protein L11 mutations in two functional domains equally affect release factors 1 and 2 activityMOLECULAR MICROBIOLOGY, Issue 1 2006Hanae Sato Summary Bacterial release factors (RFs) 1 and 2 catalyse translation termination at UAG/UAA and UGA/UAA stop codons respectively. It has been shown that limiting the amount of ribosomal protein L11 affects translation termination at UAG and UGA differently. To understand the functional interplay between L11 and RF1/RF2, we isolated 21 distinct mutations in L11 as suppressors of either temperature-sensitive (ts) RF1/RF2 strains or read-through mutants of lacZ nonsense (UAG or UGA) strains. 10 of 21 mutants restored ts lethal growth of RF1 and/or RF2 strains. All the selected L11 mutants, including the RF1ts- and RF2ts-specific suppressors, had the same effect, either enhancing or reducing, on UAG and UGA termination efficiency in vivo. The specific properties of the selected L11 mutations remained unchanged in an RF3 deletion strain. Moreover, ribosomes absent of L11 had equally reduced activity for both RF1- and RF2-mediated peptide release in vitro. These results suggest that, unlike the previous notion, L11 has a common, cooperative role with RF1 and RF2. These L11 mutations were located on the surface of two domains of L11, and interpreted to affect the interaction between L11 and rRNA or the RFs thereby leading to the altered translation termination. [source] The Aspergillus nidulans sldIRAD50 gene interacts with bimEAPC1, a homologue of an anaphase-promoting complex subunitMOLECULAR MICROBIOLOGY, Issue 1 2005Iran Malavazi Summary The Mre11,Rad50,Nbs1 protein complex has emerged as a central component in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. We have identified Aspergillus nidulans sldI1444D mutant in a screen for dynein synthetic lethals. The sldIRAD50 gene was cloned by complementation of the sporulation deficiency phenotype of this mutant. A transversion G,C at the position 2509 (Ala-692-Pro amino acid change) in the sldI1444D mutant causes sensitivity to several DNA-damaging agents. The mutation sldI1 occurs at the CXXC hinge domain of Rad50. We have deleted part of the coiled-coil and few amino acids of the Rad50,Mre11 interaction region and assessed several phenotypic traits in this deletion strain. Besides sensitivity to a number of DNA-damaging agents, this deletion strain is also impaired in the DNA replication checkpoint response, and in ascospore viability. There is no delay of the S-phase when germlings of both sldI RAD50 and mreAMRE11 inactivation strains were exposed to the DNA damage caused by bleomycin. Transformation experiments and Southern blot analysis indicate homologous recombination is dependent on scaANBS1 function in the Mre11 complex. There are epistatic and synergistic interactions between sldI RAD50 and bimEAPC1 at S-phase checkpoints and response to hydroxyurea and UV light. Our results suggest a possible novel feature of the Mre11 complex in A. nidulans, i.e. a relationship with bimE,APC1. [source] Is the Rehydrin TrDr3 from Tortula ruralis Associated with Tolerance to Cold, Salinity, and Reduced pH?PLANT BIOLOGY, Issue 3 2005HdeD from Escherichia coli in Response to Abiotic Stress, Physiological Evaluation of the TrDr3 -Orthologue Abstract: We have employed EST analysis in the resurrection moss Tortula ruralis to discover genes that control vegetative desiccation tolerance and describe the characterization of the EST-derived cDNA TrDr3 (Tortula ruralis desiccation-stress related). The deduced polypeptide TRDR3 has a predicted molecular mass of 25.5 kDa, predicted pI of 6.7, and six transmembrane helical domains. Preliminary expression analyses demonstrate that the TrDr3 transcript ratio increases in response to slow desiccation relative to the hydrated control in both total and polysomal mRNA (mRNP fraction), which classifies TrDr3 as a rehydrin. Bioinformatic searches of the electronic databases reveal that Tortula TRDR3 shares significant similarities to the hdeD gene product (HNS-dependent expression) from Escherichia coli. The function of the HdeD protein in E. coli is unknown, but it is postulated to be involved in a mechanism of acid stress defence. To establish the role of E. coli HdeD in abiotic stress tolerance, we determined the log survival percentage from shaking cultures of wild-type bacteria and the isogenic hdeD deletion strain (,hdeD) in the presence of low temperature (28 °C), elevated NaCl (5 % (w/v)), or decreased pH (4.5), or all treatments simultaneously. The ,hdeD deletion strain was less sensitive, as compared to wild-type E. coli, in response to decreased pH (p > 0.009), and the combination of all three stresses (p > 0.0001). [source] Parallel analysis of mutant human glucose 6-phosphate dehydrogenase in yeast using PCR colonies,BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2005Joshua Merritt Abstract We demonstrate a highly parallel strategy to analyze the impact of single nucleotide mutations on protein function. Using our method, it is possible to screen a population and quickly identify a subset of functionally interesting mutants. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools, and polymerase colonies. A defined mutant human glucose-6-phosphate-dehydrogenase library was constructed which contains all possible single nucleotide missense mutations in the eight-residue glucose-6-phosphate binding peptide of the enzyme. Mutant human enzymes were expressed in a zwf1 (gene encoding yeast homologue) deletion strain of Saccharomyces cerevisiae. Growth rates of the 54 mutant strains arising from this library were measured in parallel in conditions selective for active hG6PD. Several residues were identified which tolerated no mutations (Asp200, His201 and Lys205) and two (Ile199 and Leu203) tolerated several substitutions. Arg198, Tyr202, and Gly204 tolerated only 1-2 specific substitutions. Generalizing from the positions of tolerated and non-tolerated amino acid substitutions, hypotheses were generated about the functional role of specific residues, which could, potentially, be tested using higher resolution/lower throughput methods. © 2005 Wiley Periodicals, Inc. [source] Cyclophilin D links programmed cell death and organismal aging in Podospora anserinaAGING CELL, Issue 5 2010Diana Brust Summary Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl- cis,trans -isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild-type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging. [source] SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulansMOLECULAR MICROBIOLOGY, Issue 5 2001Harald Oberegger Under conditions of low iron availability, most fungi excrete siderophores in order to mobilize extracellular iron. We show that lack of the GATA-type transcription factor SREA in Aspergillus nidulans not only leads to derepression of siderophore biosynthesis but also to deregulation of siderophore-bound iron uptake and ornithine esterase expression. Furthermore, SREA deficiency causes increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA deletion strains, extracellular siderophore production is derepressed but still regulated negatively by iron availability, indicating the presence of an additional iron-regulatory mechanism. In contrast, iron affects ferricrocin accumulation in a positive way, suggesting a protective role for this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes catalase CATB and the superoxide dismutases SODA and SODB. It is noteworthy that iron starvation was found to repress catB expression in wild-type (wt) and SREA-deficient strains, consistent with catB being subject to SREA-independent iron regulation. Differential display led to the identification of putative SREA target genes amcA and mirA. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. amcA encodes a putative mitochondrial carrier for the siderophore precursor ornithine, indicating cross-regulation of siderophore and ornithine metabolism. [source] Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruitsMOLECULAR PLANT PATHOLOGY, Issue 3 2008I. MIYARA SUMMARY Tissue alkalinization during Colletotrichum gloeosporioides attack enhances the expression of PELB, which encodes pectate lyase (PL), and PL secretion, which is considered essential for full virulence. We studied the regulation of PL secretion by manipulation of C. gloeosporioides PELB. PELB was down-regulated by knocking out PAC1, which encodes the PacC transcription factor that regulates gene products with pH-sensitive activities. We functionally characterized a PACC gene homologue, PAC1, from C. gloeosporioides wild-type (WT) Cg-14 and two independent deletion strains, ,pac1372and ,pac1761. Loss-of-function PAC1 mutants showed 85% reduction of PELB transcript expression, delayed PL secretion and dramatically reduced virulence, as detected in infection assays with avocado fruits. In contrast, PELB was up-regulated in the presence of carbon sources such as glucose. When glucose was used as a carbon source in the medium for the WT strain and the ,pac1 mutant at pH 6.0, PELB transcript expression and PL secretion were activated. Other sugars, such as sucrose and fructose (but not galactose), also activated PELB expression. These results suggest that the pH-regulated response is only part of a multi-factor regulation of PELB, and that sugars are also needed to promote the transition from quiescent to active necrotrophic development by the pathogen. [source] | ||||||||||