bluuebunny/crossref_metadata_2025_split huggingface dataset:
Opi1p, Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade
Authors: Kaadige Mohan R. , Lopes John M. 
Published: December 2004

SummaryThe INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes. INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p‐mediated repression and Ume6p‐mediated activation. To screen for mutants with altered INO2 expression directly, we constructed an INO2‐HIS3 reporter that provides a plate assay for INO2 promoter activity. This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild‐type strain. The dim1 mutants contain mutations in the OPI1 gene. To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Δ mutation that overexpress the INO2‐HIS3 gene. Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Δ mutation. These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes. This prompted us to examine the effect of sin3Δ and rpd3Δ mutants on INO2‐cat expression. Surprisingly, the sin3Δ allele overexpressed INO2‐cat, whereas the rpd3Δ mutant had no effect. We also show that the UME6 gene does not affect the expression of an OPI1‐cat reporter. This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression.
Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis
Authors: Park Heui‐Dong , Guinn Kristi M. , Harrell Maria I. , Liao Reiling , Voskuil Martin I. , Tompa Martin , Schoolnik Gary K. , Sherman David R. 
Published: December 2004

SummaryUnlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild‐type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha‐crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence‐based predictions that the C‐terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two‐component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.
Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs
Authors: Yin Zhikang , Wilson Séan , Hauser Nicole C. , Tournu Helene , Hoheisel Jörg D. , Brown Alistair J. P. 
Published: December 2004

SummaryGlucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post‐transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.
Identification of a novel plasmin(ogen)‐binding motif in surface displayed α‐enolase of Streptococcus pneumoniae
Authors: Bergmann Simone , Wild Daniela , Diekmann Oliver , Frank Ronald , Bracht Dagmar , Chhatwal Gursharan S. , Hammerschmidt Sven 
Published: December 2004

SummaryThe interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface‐associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal α‐enolase (Eno) and is subsequently activated to the serine protease plasmin by host‐derived tissue plasminogen activator (tPA) or urokinase (uPA). The C‐terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)‐binding site of Eno by investigating the protein–protein interaction. Plasmin(ogen)‐binding activity of C‐terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno‐plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248–256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)‐binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing α‐enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen‐binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen‐binding motif in the pathogenesis of pneumococcal diseases.
SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development
Authors: Dessens Johannes T. , Sidén‐Kiamos Inga , Mendoza Jacqui , Mahairaki Vassiliki , Khater Emad , Vlachou Dina , Xu Xiao‐Jin , Kafatos Fotis C. , Louis Christos , Dimopoulos George , Sinden Robert E. 
Published: December 2004

SummaryAn essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite‐producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete‐to‐oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine‐rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast‐two‐hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete‐to‐oocyst differentiation in mosquitoes.
An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4‐activated 6‐phosphofructokinase
Authors: Nariya Hirofumi , Inouye Sumiko 
Published: December 2004

Summary6‐Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7‐fold upon phosphorylation at Thr‐226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk–pkn4 deletion strain accumulated glycogen at a higher level than the wild‐type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk–pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr‐226‐Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.
Surface anchoring of bacterial subtilisin important for maturation function
Authors: Coutte Loic , Willery Eve , Antoine Rudy , Drobecq Hervé , Locht Camille , Jacob‐Dubuisson Françoise 
Published: December 2004

SummaryMany extracytoplasmic proteins undergo proteolytic processing during secretion, which is essential to their maturation. These post‐translational modifications are carried out by specific enzymes whose subcellular localization is important for function. We have described a maturation subtilisin in Gram‐negative Bordetella pertussis, the autotransporter SphB1. SphB1 catalyses the maturation of the precursor of the adhesin filamentous haemagglutinin (FHA) at the bacterial surface, in addition to the processing of its own precursor. Here, we show that the outer membrane anchor of SphB1 is crucial to its function, as evidenced by the lack of FHA maturation in a strain releasing a variant of SphB1 into the milieu. In contrast, surface association is not required for automaturation of SphB1. The surface retention of mature SphB1 is mediated by lipidation of the protein. The tethered protease appears to be stabilized by unusual Gly‐ and Pro‐rich motifs at the N‐terminus of the protein. This represents a new mode of localization for a protease involved in protein secretion.
Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner
Authors: Youderian Philip , Burke Neal , White David J. , Hartzell Patricia L. 
Published: December 2004

Summary Myxococcus xanthus glides over solid surfaces without the use of flagella, dependent upon two large sets of adventurous (A) and social (S) genes, using two different mechanisms of gliding motility. Myxococcus xanthus A–S– double mutants form non‐motile colonies lacking migratory cells at their edges. We have isolated 115 independent mutants of M. xanthus with insertions of transposon magellan‐4 in potential A genes by screening for insertions that reduce the motility of a mutant S– parental strain. These insertions are found not only in the three loci known to be required for A motility, mglBA, cglB, and aglU, but also in 30 new genes. Six of these new genes encode different homologues of the TolR, TolB, and TolQ transport proteins, suggesting that adventurous motility is dependent on biopolymer transport. Other insertions which affect both A and S motility suggest that both systems share common energy and cell wall determinants. Because the spectrum of magellan‐4 insertions in M. xanthus is extraordinarily broad, transposon mutagenesis with this eukaryotic genetic element permits the rapid genetic analysis of large sets of genes that contribute to a complex microbial behaviors such as A motility.
Invasive M1T1 group A Streptococcus undergoes a phase‐shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB
Authors: Aziz Ramy K. , Pabst Michael J. , Jeng Arthur , Kansal Rita , Low Donald. E. , Nizet Victor , Kotb Malak 
Published: December 2004

SummaryA globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2‐D gel analysis of the secreted M1T1 proteome, coupled with MALDI‐TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA– M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase‐shift to a SpeB–/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte‐stimulating capacity. The proteome of the SpeB–in vivo phase‐shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage‐encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB– phase‐shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.
Characterization of components of the mismatch repair machinery in Trypanosoma brucei
Authors: Bell Joanna S. , Harvey Timothy I. , Sims Anne‐Marie , McCulloch Richard 
Published: December 2004

SummaryMismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base–base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.
Authentic precursors to ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone
Authors: Hage Aziz El , Alix Jean‐Hervé 
Published: December 2004

Summary Escherichia coli dnaK‐ts mutants are defective in the late stages of ribosome biogenesis at high temperature. Here, we show that the 21S, 32S and 45S ribosomal particles that accumulate in the dnaK756‐ts mutant at 44°C contain unprocessed forms of their 16S and 23S rRNAs (partially processed in the case of 45S particles). Their 5S rRNA stoichiometry and ribosomal protein composition are typical of the genuine ribosomal precursors found in a wild‐type (dnaK+) strain. Despite the lack of a functional DnaK, a very slow maturation of these 21S, 32S and 45S particles to structurally and functionally normal 30S and 50S ribosomal subunits still occurs at high temperature. This conversion is accompanied by the processing of p16S and p23S rRNAs to their mature forms. We conclude that: (i) 21S, 32S and 45S particles are not dead‐end particles, but true precursors to active ribosomes (21S particles are converted to 30S subunits, and 32S and 45S to 50S subunits); (ii) DnaK is not absolutely necessary for ribosome biogenesis, but accelerates the late steps of this process considerably at high temperature; and (iii) 23S rRNA processing depends on the stage reached in the stepwise assembly of the 50S subunit, not directly on DnaK.
Evidence for dynamic clustering of carboxy‐terminal aromatic amino acids in TonB‐dependent energy transduction
Authors: Ghosh Joydeep , Postle Kathleen 
Published: December 2004

Summary Escherichia coli uses the proton motive force of the cytoplasmic membrane and TonB protein to energize the active transport of iron‐siderophores and vitamin B12 across the outer membrane. TonB shuttles between the cytoplasmic and outer membranes, presumably during the course of energy transduction. Previous results indicated that the carboxy‐terminal 65 amino acids of TonB are essential for both its outer membrane association and activity. A highly conserved region (residues 199–216) within this domain, predicted to be an amphipathic α‐helix, was the initial focus of this study. Scanning mutagenesis indicated that only the aromatic residues F202, W213 and Y215 were individually important for activity. When the crystal structure of a dimeric TonB carboxy‐terminus subsequently became available, we observed that two additional aromatic residues outside that region, F180 and F230, were potentially engaged in end‐on hydrophobic interactions with the three residues identified previously. Changing these five aromatic residues individually to alanine reduced TonB activity. Surprisingly, however, each substitution exhibited a unique phenotypic profile with respect to ability to support [55Fe]‐ferrichrome transport, sensitivity to colicins B, D, Ia and M or sensitivity to bacteriophage φ80. The phenotypic results suggested that the carboxy‐terminus of TonB was a flexible and dynamic domain that could interact specifically with different ligands or transporters, perhaps through the aromatic residues. The possibility of interactions among all the aromatic residues was tested using double‐mutant cycle analysis. All possible combinations of alanine substitutions were constructed, with the result that TonB containing any double‐alanine substitution was inactive in the phenotypic assays, while retaining the ability to associate with the outer membrane. This synergistic, rather than additive, effect of the double mutants suggested that, consistent with the flexibility suggested by analysis of the single substitutions, all the aromatic residues might be capable of interacting with one another. A means of reconciling these results with the crystal structure is presented.
Characterization of a non‐mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum
Authors: Baunaure Françoise , Eldin Patrick , Cathiard Anne‐Marie , Vial Henri 
Published: December 2004

SummaryIn search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite‐encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum‐infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl‐terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 ± 19 µM and a VMAX of 680 ± 49 nmol of phosphatidylethanolamine formed h−1 mg−1 protein. Site‐directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non‐identical subunits (α and β) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co‐localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.
The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury
Authors: Kwok Lai Yu , Schlüter Dirk , Clayton Christine , Soldati Dominique 
Published: December 2004

SummarySuperoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2‐detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin‐like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1‐cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.