bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE
Authors: Cava Felipe , Laptenko Oleg , Borukhov Sergei , Chahlafi Zahra , Blas‐Galindo Emilio , Gómez‐Puertas Paulino , Berenguer José 
Published: May 2007

SummaryThe strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc‐NADH dehydrogenase and the Nar‐nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen‐sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase σA holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.
An antisense RNA controls synthesis of an SOS‐induced toxin evolved from an antitoxin
Authors: Kawano Mitsuoki , Aravind L. , Storz Gisela 
Published: May 2007

SummaryOnly few small, regulatory RNAs encoded opposite another gene have been identified in bacteria. Here, we report the characterization of a locus where a small RNA (SymR) is encoded in cis to an SOS‐induced gene whose product shows homology to the antitoxin MazE (SymE). Synthesis of the SymE protein is tightly repressed at multiple levels by the LexA repressor, the SymR RNA and the Lon protease. SymE co‐purifies with ribosomes and overproduction of the protein leads to cell growth inhibition, decreased protein synthesis and increased RNA degradation. These properties are shared with several RNA endonuclease toxins of the toxin‐antitoxin modules, and we show that the SymE protein represents evolution of a toxin from the AbrB fold, whose representatives are typically antitoxins. We suggest that SymE promotion of RNA cleavage may be important for the recycling of RNAs damaged under SOS‐inducing conditions.
Whole‐genome analysis of the chromosome partitioning and sporulation protein Spo0J (ParB) reveals spreading and origin‐distal sites on the Bacillus subtilis chromosome
Authors: Breier Adam M. , Grossman Alan D. 
Published: May 2007

SummaryWe investigated the genome‐wide DNA binding of the chromosome partitioning and sporulation protein and ParB family member Spo0J in Bacillus subtilis using chromatin immunoprecipitation and DNA microarrays. We identified 10 parS loci to which Spo0J binds, two of which were unexpectedly distant (> 1 Mb) from the origin of replication. We used all 10 sites to refine the consensus sequence for parS. We found that Spo0J spreads along the DNA around each site. Binding was near maximal levels up to 1.6 kb away from parS, and significantly above background as far away as 18 kb. Deletion of soj (parA) had little or no effect on spreading. In contrast, the spo0J93 allele appeared to cause a significant decrease in spreading in vivo, without significantly affecting the DNA binding affinity in vitro. spo0J93 causes a phenotype similar to that of a spo0J null mutant and alters the region thought to be involved in interaction between Spo0J dimers. Our findings indicate that spreading is important for in vivo function of Spo0J. Gene expression in areas near parS sites was similar in wild type and a spo0J null mutant, indicating that binding and spreading of Spo0J on DNA does not normally silence transcription of nearby genes.
Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains
Authors: Tourand Yvonne , Lee Linda , Chaconas George 
Published: May 2007

Summary Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C‐terminal domain (ResT164−449) displays sequence‐specific DNA binding to the box 3,4,5 region of the telomere, while the N‐terminal domain (ResT1−163) exhibits sequence‐independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N‐terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT164−449. Conversely, ResT1−164 may play an important regulatory role by modulating both sequence‐specific DNA binding activity and catalysis by the C‐terminal domain.
The RecJ DNase strongly suppresses genomic integration of short but not long foreign DNA fragments by homology‐facilitated illegitimate recombination during transformation of Acinetobacter baylyi
Authors: Harms Klaus , Schön Verena , Kickstein Eva , Wackernagel Wilfried 
Published: May 2007

SummaryHomology‐facilitated illegitimate recombination (HFIR) promotes genomic integration of foreign DNA with a single segment homologous to the recipient genome by homologous recombination in the segment accompanied by illegitimate fusion of the heterologous sequence. During natural transformation of Acinetobacter baylyi HFIR occurs at about 0.01% of the frequency of fully homologous recombination. The role of the 5′ single‐strand‐specific exonuclease RecJ in HFIR was investigated. Deletion of recJ increased HFIR frequency about 20‐fold compared with wild type while homologous recombination was not affected. Illegitimate fusion sites were predominantly located within 360 nucleotides away from the homology whereas in wild type most fusion sites were distal (500–2500 nucleotides away). RecJ overproduction reduced the HFIR frequency to half compared with wild type, and transformants with short foreign DNA segments were diminished, leading to on average 866 foreign nucleotides integrated per event (682 in wild type, 115 in recJ). In recJ always the 3′ ends of donor DNA were integrated at the homology whereas in wild type these were 3′ or 5′. RecJ apparently suppresses HFIR by degrading 5′ non‐homologous DNA tails at the post‐synaptic stage. We propose that the RecJ activity level controls the HFIR frequency during transformation and the amount of foreign DNA integrated per event.
Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence
Authors: Moyrand Frédérique , Fontaine Thierry , Janbon Guilhem 
Published: May 2007

SummaryThe polysaccharidic capsule is the main virulence factor of Cryptococcus neoformans. It primarily comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and galactoxylomannan (GalXM, 7% of the capsule mass). We constructed a large collection of mutant strains in which genes potentially involved in capsule biosynthesis were deleted. We used a new post‐genomic approach to study the virulence of the strains. Primers specific for unique tags associated with the disruption cassette were used in a real‐time PCR virulence assay to measure the fungal burden of each strain in different organs of mice in multi‐infection experiments. With this very sensitive assay, we identified a putative UDP‐glucose epimerase (Uge1p) and a putative UDP‐galactose transporter (Ugt1p) essential for C. neoformans virulence. The uge1Δ and ugt1Δ strains are temperature sensitive and do not produce GalXM but synthesize a larger capsule. These mutant strains (GalXM negative, GXM positive) are not able to colonize the brain even at the first day of infection whereas GXM‐negative strains (GalXM positive) can still colonize the brain, although less efficiently than the wild‐type strain.
Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus
Authors: Zolghadr Behnam , Weber Stefan , Szabó Zalán , Driessen Arnold J. M. , Albers Sonja‐Verena 
Published: May 2007

SummaryThe hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracellular macromolecular surface appendages. Likewise, S. solfataricus binding proteins have been suggested to assemble in higher ordered surface structures as well, tentatively termed the bindosome. Here we show that S. solfataricus contains a specific system that is needed for the functional surface localization of sugar binding proteins. This system, encoded by the bas (bindosome assembly system) operon, is composed of five proteins: basABC, three homologues of so‐called bacterial (pseudo)pilins; BasE, a cytoplasmic ATPase; and BasF, an integral membrane protein. Deletion of either the three (pseudo)pilin genes or the basEF genes resulted in a severe defect of the cells to grow on substrates which are transported by sugar binding proteins containing class III signal peptides, while growth on glucose and maltose was restored when the corresponding genes were reintroduced in these cells. Concomitantly, ΔbasABC and ΔbasEF cells were severely impaired in glucose uptake even though the sugar binding proteins were normally secreted across the cytoplasmic membrane. These data underline the hypothesis that the bas operon is involved in the functional localization of sugar binding proteins at the cell surface of S. solfataricus. In contrast to surface structure assembly systems of Gram‐negative bacteria, the bas operon seems to resemble an ancestral simplified form of these machineries.
Split target specificity of ResT: a design for protein delivery, site selectivity and regulation of enzyme activity?
Authors: Jayaram Makkuni 
Published: May 2007

SummaryThe ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N‐terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N‐ and C‐terminal domains targeting distinct regions of the telomere. This bi‐specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.
Submicromolar hydrogen peroxide disrupts the ability of Fur protein to control free‐iron levels in Escherichia coli
Authors: Varghese Shery , Wu Amy , Park Sunny , Imlay Karin R. C. , Imlay James A. 
Published: May 2007

SummaryIn aerobic environments, mutants of Escherichia coli that lack peroxidase and catalase activities (Hpx–) accumulate submicromolar concentrations of intracellular H2O2. We observed that in defined medium these strains constitutively expressed members of the Fur regulon. Iron‐import proteins, which Fur normally represses, were fully induced. H2O2 may antagonize Fur function by oxidizing the Fur:Fe2+ complex and inactivating its repressor function. This is a potential problem, as in iron‐rich environments excessive iron uptake would endanger H2O2‐stressed cells by accelerating hydroxyl‐radical production through the Fenton reaction. However, the OxyR H2O2‐response system restored Fur repression in iron‐replete Luria–Bertani medium by upregulating the synthesis of Fur protein. Indeed, when the OxyR binding site upstream of fur was disrupted, Hpx– mutants failed to repress transporter synthesis, and they exhibited high levels of intracellular free iron. Mutagenesis and bacteriostasis resulted. These defects were eliminated by mutations or chelators that slowed iron import, confirming that dysregulation of iron uptake was the root problem. Thus, aerobic organisms must grapple with a conundrum: how to monitor iron levels in oxidizing environments that might perturb the valence of the analyte. The induction of Fur synthesis by the OxyR response comprises one evolutionary solution to that problem.
Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin
Authors: Santos Antonio , San Mauro Manuel , Abrusci Concepción , Marquina Domingo 
Published: May 2007

SummaryPMKT is a channel‐forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2Δ mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT‐affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring‐defective mutants were much less sensitive to PMKT, as were wild‐type protoplasts pretreated with phosphatidylinositol‐specific phospholipase C to remove GPI‐anchored proteins, indicating that the GPI‐anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein‐entrapped liposomes containing a purified GFP–Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein‐free liposomes. Cwp2p and its GPI‐anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.
Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB
Authors: Mitchell Jennifer , Siboo Ian R. , Takamatsu Daisuke , Chambers Henry F. , Sullam Paul M. 
Published: May 2007

SummaryPblA and PblB are prophage‐encoded proteins of Streptococcus mitis strain SF100 that mediate binding to human platelets. The mechanism for surface expression of these proteins has been unknown, as they do not contain signal sequences or cell wall sorting motifs. We therefore assessed whether expression of these proteins was linked the lytic cycle of the prophage. Deletion of either the holin or lysin gene resulted in retention of PblA and PblB in the cytoplasm, and loss of these proteins from the cell wall. Flow cytometric analysis revealed that induction of phage replication in SF100 produced a subpopulation of cells with increased permeability. This effect was abrogated by disruption of the holin and lysin genes. Treatment of these mutants with exogenous PblA and PblB restored surface expression, apparently via binding of the proteins to cell wall choline. Loss of PblA and PblB expression was associated with decreased platelet binding in vitro, and reduced virulence in an animal model of endocarditis. Thus, expression of PblA and PblB occurs via a novel mechanism, whereby phage induction increases bacterial permeability and release of the proteins, followed by their binding to surface of viable cells. This mechanism may be important for endovascular infection.
Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress
Authors: Van Den Bogaart Geert , Hermans Nicolaas , Krasnikov Victor , Poolman Bert 
Published: May 2007

SummaryThe effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo‐bleaching, pulsed‐FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed‐FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 μm2 s−1, whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D∼0.4 μm2 s−1). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.
The shape of galaxy cluster dark matter haloes: systematics of its imprint on cluster gas and comparison to observations
Authors: Flores Ricardo A. , Allgood Brandon , Kravtsov Andrey V. , Primack Joel R. , Buote David A. , Bullock James S. 
Published: May 2007

Abstract
We study predictions for galaxy cluster observables that can test the statistics of dark matter halo shapes expected in a flat Λ cold dark matter (CDM) universe. We present a simple analytical model for the prediction of cluster-scale X-ray observations, approximating clusters as isothermal systems in hydrostatic equilibrium, and dark matter haloes as ellipsoids with uniform axial ratios (homeoidal ellipsoids). We test the model against high-resolution, hydrodynamic cluster simulations to gauge its reliability. We find that this simple prescription does a good job of predicting cluster X-ray ellipticities compared to the simulations as long as one focuses on cluster regions that are less sensitive to recent mergers. Based on this simple model, the distribution of cluster-size halo shapes expected in the concordance ΛCDM cosmology implies an X-ray ellipticity distribution with a mean 〈εX〉= 0.32 ± 0.01, and a scatter σε= 0.14 ± 0.01 for the mass range (1–4) × 1014h−1 M⊙. We find it important to include the mass dependence of halo shape when making comparisons to observational samples. We analyse the systematics of four observational samples of cluster ellipticities and find that our results are statistically compatible with these observations. In particular, we find remarkably good agreement between two recent ROSAT samples and ΛCDM predictions that do not include gas cooling. We also test how well our analytical model can predict Sunyaev–Zel'dovich decrement maps and find that it is less successful although still useful; the model does not perform as well as a function of flux level in this case because of the changing triaxiality of dark matter haloes as a function of radial distance. Both this effect and the changing alignment of isodensity shells of dark matter haloes leave an imprint on cluster gas that appears to be seen in observational data. Thus, dark matter haloes cannot be accurately characterized as homeoidal ellipsoids for all comparisons.
Mechanism of CO2 acquisition in an acid‐tolerant Chlamydomonas
Authors: BALKOS KONSTANTINE D. , COLMAN BRIAN 
Published: May 2007

ABSTRACTThe acid‐tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using 14C‐benzoic acid (BA) or [2‐14C]‐5,5‐dimethyloxazolidine‐2,4‐dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0–7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO2 uptake and O2 evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO2 uptake. The CO2 compensation concentration of the cells ranged from 9.4 µM at pH 4.5 to 16.2 µM at pH 7.0. An examination of the kinetics of ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the Km (CO2) was 16.3 µM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO2, and the expression of external CA at neutral pH values would maintain an equilibrium CO2 concentration at the cell surface. This species does not possess a CO2‐concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low Km (CO2) Rubisco of the alga.
The stomatal response to evaporative demand persists at night in Ricinus communis plants with high nocturnal conductance
Authors: BARBOUR MARGARET M. , BUCKLEY THOMAS N. 
Published: May 2007

ABSTRACTEvidence is building that stomatal conductance to water vapour (gs) can be quite high in the dark in some species. However, it is unclear whether nocturnal opening reflects a mechanistic limitation (i.e. an inability to close at night) or an adaptive response (i.e. promoting water loss for reasons unrelated to carbon gain). Further, it is unclear if stomatal responses to leaf‐air vapour pressure difference (D) persist in the dark. We investigated nocturnal stomatal behaviour in castor bean (Ricinus communis L.) by measuring gas exchange and stomatal responses to D in the light and in the dark. Results were compared among eight growth environments [two levels for each of three treatment variables: air saturation deficit (Da), light and water availability]. In most plants, stomata remained open and sensitive to D at night. gs was typically lower at night than in the day, whereas leaf osmotic pressure (Π) was higher at night. In well‐watered plants grown at low Da, stomata were less sensitive to D in the dark than in the light, but the reverse was found for plants grown at high Da. Stomata of droughted plants were less sensitive to D in the dark than in the light regardless of growth Da. Drought also reduced gs and elevated Π in both the light and the dark, but had variable effects on stomatal sensitivity to D. These results are interpreted with the aid of models of stomatal conductance.