bluuebunny/crossref_metadata_2025 huggingface dataset:
Electromigration induced ductile-to-brittle transition in lead-free solder joints
Authors: Ren Fei , Nah Jae-Woong , Tu K. N. , Xiong Bingshou , Xu Luhua , Pang John H. L.
Published: October 2006

The effect of electromigration on ductile-to-brittle transition in flip chip solder joints has been studied using one-dimensional bamboo-type samples of eutectic 95.5Sn–3.8Ag–0.7Cu solder joined by Cu wires at two ends. Both electrical current and tensile stress were applied to the samples either in serial or in parallel. In serial tests, the strain rate was 6×10−3. In parallel test, the creep stress was 7MPa. The current density applied was (1–5)×103A∕cm2. The working temperature was 100–150°C. In both tests, the authors observed the ductile-to-brittle transition in which the fracture migrates from the middle to the cathode interface of the joint with increasing current density and time. The transition is explained by the polarity effect of electromigration, especially the accumulation of vacancies at the cathode interface.
Tip-enhanced fluorescence microscopy of high-density samples
Authors: Xie Changan , Mu Chun , Cox Jonathan R. , Gerton Jordan M.
Published: October 2006

High-density samples of fluorescent quantum dots (QDs) were imaged using an apertureless near-field optical microscopy technique. QD fluorescence was modulated by oscillating a silicon atomic force microscope tip above an illuminated sample and a lock-in amplifier was used to suppress background from the excitation laser. Spatial resolution near 10nm and a peak signal-to-noise ratio (SNR) of ∼60 were achieved. Individual QDs within high-density ensembles were still easily resolved (SNR>5) at a density of 14QDs∕μm2. These results have favorable implications for the eventual nanoscale imaging of viable biological systems, such as cellular membranes.
Chemical states and electrical properties of a high-k metal oxide/silicon interface with oxygen-gettering titanium-metal-overlayer
Authors: Seo Kang-Ill , Lee Dong-Ick , Pianetta Piero , Kim Hyoungsub , Saraswat Krishna C. , McIntyre Paul C.
Published: October 2006

The authors report on the chemical bonding structure of the HfO2∕Si (001) stack after the SiO2 interfacial layer (IL) is partially removed by a reactive titanium metal overlayer. Using synchrotron photoelectron spectroscopy, they found that ultrathin SiO2-like IL ∼6.5Å thick, which is significantly less than the initial SiO2 IL thickness of ∼15Å, exists at the HfO2∕Si interface with an overlying Ti electrode. The dissociated Si from SiO2 IL is believed to go onto Si substrate where it regrows epitaxially. The interfacial trap density of the Ti-electrode sample was extracted to be ∼1.6×1011eV−1cm−2 near the midgap of Si, which was comparable to that of the control sample with W electrode.
Inverted bulk-heterojunction organic photovoltaic device using a solution-derived ZnO underlayer
Authors: White M. S. , Olson D. C. , Shaheen S. E. , Kopidakis N. , Ginley D. S.
Published: October 2006

Inverted organic photovoltaic devices based on a blend of poly(3-hexylthiophene) and a fullerene have been developed by inserting a solution-processed ZnO interlayer between the indium tin oxide (ITO) electrode and the active layer using Ag as a hole-collecting back contact. Efficient electron extraction through the ZnO and hole extraction through the Ag, with minimal loss in open-circuit potential, is observed with a certified power conversion efficiency of 2.58%. The inverted architecture removes the need for the use of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) as an ITO modifier and for the use of a low-work-function metal as the back contact in the device.
Effect of CaRuO3 interlayer on the dielectric properties of Ba(Zr,Ti)O3 thin films prepared by pulsed laser deposition
Authors: Tang X. G. , Tian H. Y. , Wang J. , Wong K. H. , Chan H. L. W.
Published: October 2006

Ba ( Zr 0.2 Ti 0.8 ) O 3 (BZT) thin films on Pt(111)∕Ti∕SiO2∕Si(100) substrates without and with CaRuO3 (CRO) buffer layer were fabricated at 650°C in situ by pulsed laser deposition. The BZT thin films showed a dense morphology, many clusters are found on the surface images of BZT/Pt films, which are composed by nanosized grains of 25–35nm; the average grain size of BZT/CRO films is about 80nm, which lager than that of BZT/Pt thin film. The dielectric constants and dissipation factors of BZT/Pt and BZT/CRO thin films were 392 and 0.019 and 479 and 0.021 at 1MHz, respectively. The dielectric constant of BZT/Pt and BZT/CRO thin films changes significantly with applied dc bias field and has high tunabilities and figures of merit of ∼70% and 37 and 75% and 36, respectively, under an applied field of 400kV∕cm. The possible microstructural background responsible for the high dielectric constant and tunability was discussed.
Detection of phospholipid-carbon nanotube translocation using fluorescence energy transfer
Authors: Lin Sijie , Keskar Gayatri , Wu Yonnie , Wang Xi , Mount Andrew S. , Klaine Stephen J. , Moore Jessica M. , Rao Apparao M. , Ke Pu Chun
Published: October 2006

Single-walled carbon nanotubes (SWNTs) and lysophospholipids readily assemble into supramolecular complexes in aqueous solutions. Upon light excitation the fluorescence of rhodamine-labeled lysophospholipids was redshifted and quenched due to the optical absorption of the SWNTs. Utilizing fluorescence energy transfer, the authors detected the translocation and disassembly of SWNT complexes in MCF breast cancer cells. These lipid-coated SWNT complexes enable drugs to be delivered at an effective dose and their subsequent release to be monitored in real time.
Predicted climate change alters the indirect effect of predators on an ecosystem process
Authors: Lensing Janet R. , Wise David H.
Published: October 2006

Changes in rainfall predicted to occur with global climate change will likely alter rates of leaf-litter decomposition through direct effects on primary decomposers. In a field experiment replicated at two sites, we show that altered rainfall may also change how cascading trophic interactions initiated by arthropod predators in the leaf litter indirectly influence litter decomposition. On the drier site there was no interaction between rainfall and the indirect effect of predators on decomposition. In contrast, on the moister site spiders accelerated the disappearance rate of deciduous leaf litter under low rainfall, but had no, or possibly a negative, indirect effect under high rainfall. Thus, changes resulting from the more intense hydrological cycle expected to occur with climate change will likely influence how predators indirectly affect an essential ecosystem process.
Smoking, healthcare cost, and loss of productivity in Sweden 2001
Authors: Bolin Kristian , Lindgren Björn
Published: October 2006

Aims: Objectives were (a) to estimate healthcare cost and productivity losses due to smoking in Sweden 2001 and (b) to compare the results with studies for Sweden 1980, Canada 1991, Germany 1996, and the USA 1998. Methods: Published estimates on relative risks and Swedish smoking patterns were used to calculate attributable risks for smokers and former smokers. These were applied to cost estimates for smoking-related diseases based on data from public Swedish registers. Results: The estimated total cost for Sweden 2001 was US$ 804 million; COPD and cancer of the lung accounted for 43%. Healthcare cost accounted for 26% of the total cost. The estimated costs per smoker were US$ 3,200 in the USA 1998; 1,600 in Canada 1991; 1,100 in Germany 1996; 600 in Sweden 2001; and 300 in Sweden 1980 (all in 2001 US$ prices). Conclusions: To reduce the prevalence of smoking is an issue worthwhile pursuing in its own right. In order to reduce the cost of smoking, however, policy-makers should also explore and influence the factors that determine the cost per smoker. Sweden seems to have been more successful than comparable countries in pursuing both these objectives.
Identification and characterization of arginase II as a chondrocyte phenotype‐specific gene
Authors: Lee Hyun‐Chae , Song Woo‐Keun , Lee Young‐Sup , Chun Jang‐Soo
Published: October 2006

We have previously shown that activation of extracellular signal‐regulated protein kinase‐1 and ‐2 (ERK1/2) causes chondrocyte dedifferentiation, which contributes to the destruction of arthritic cartilage. In the present study, we identified genes involved in the ERK1/2 regulation of chondrocyte dedifferentiation. Several genes were identified by subtractive hybridization, and, of these, arginase II was selected for further functional characterization. Similar to the pattern of type II collagen expression, which is a hallmark of chondrocyte differentiation, arginase II expression was increased during chondrogenesis of mesenchymal cells. The high expression level of arginase II was decreased during dedifferentiation of chondrocytes, whereas its expression was restored during redifferentiation of the dedifferentiated chondrocytes. Inhibition of ERK1/2 signaling in chondrocytes enhanced type II collagen expression with a concomitant increase in expression and activity of arginase II. However, ectopic expression of arginase II or inhibition of its activity did not affect chondrocyte differentiation. The results collectively indicate that expression of arginase II is specific to the chondrocyte phenotype, although the expression of arginase II alone is not sufficient for articular chondrocytes to maintain a differentiated phenotype.
iubmb Life, 58: 597‐605, 2006
RNA damage and surveillance under oxidative stress
Authors: Li Zhongwei , Wu Jinhua , DeLeo Christopher J.
Published: October 2006

RNA damage has been recently reported to increase under oxidative stress and in patients with many degenerative diseases, which has drawn attention to the consequences of RNA oxidation at the molecular and cellular levels. Under similar conditions the levels of oxidative damage in RNA are usually higher than those in DNA, which may impair protein synthesis or other RNA function. Therefore, accumulation of RNA damage must be prevented and cells have developed specific mechanisms to remove oxidatively‐damaged RNA and to block incorporation of oxidized nucleotides during RNA synthesis. Removal of oxidized RNA may be mediated by specific proteins that recognize oxidative lesions and direct the RNA degradation machinery to eliminate the damaged RNAs. During RNA synthesis, oxidized ribonucleotides are hydrolyzed or discriminated from normal ribonucleotides during transcription, preventing their incorporation into RNA. Collective evidence suggests that RNA oxidative damage is a challenging and persistent problem normally controlled through RNA surveillance mechanisms, making them critical to maintaining cellular health and preventing disease.
iubmb Life, 58: 581‐588, 2006
Nepotism and brood reliability in the suppression of worker reproduction in the eusocial Hymenoptera
Authors: Nonacs Peter
Published: October 2006

In many eusocial Hymenoptera, workers prevent each other from producing male offspring by destroying worker-laid eggs. Kin selection theory predicts that such ‘worker policing’ behaviour can evolve by increasing the average relatedness between workers and their male brood. Alternatively, if worker-laid eggs are of low relative viability, their replacement would increase the developmental reliability of the brood. Less colony investment in terms of time and resources would be lost on poor males. This gain is independent of the relatedness of the males. Unfortunately, both nepotistic and group efficiency benefits can simultaneously accrue with the replacement of worker-laid eggs. Therefore, worker behaviour towards eggs cannot completely resolve whether both processes have been equally evolutionarily important. Adequate resolution requires the presentation of worker-produced brood of various ages. The stage at which brood are replaced can discriminate whether worker policing occurs owing to a preference for closer genetic kin, a preference for the more reliable brood or both.
Healing substrates with mobile, particle-filled microcapsules: designing a ‘repair and go’ system
Authors: Verberg Rolf , Dale Alex T , Kumar Prashant , Alexeev Alexander , Balazs Anna C
Published: October 2006

We model the rolling motion of a fluid-driven, particle-filled microcapsule along a heterogeneous, adhesive substrate to determine how the release of the encapsulated nanoparticles can be harnessed to repair damage on the underlying surface. We integrate the lattice Boltzmann model for hydrodynamics and the lattice spring model for the micromechanics of elastic solids to capture the interactions between the elastic shell of the microcapsule and the surrounding fluids. A Brownian dynamics model is used to simulate the release of nanoparticles from the capsule and their diffusion into the surrounding solution. We focus on a substrate that contains a damaged region (e.g. a crack or eroded surface coating), which prevents the otherwise mobile capsule from rolling along the surface. We isolate conditions where nanoparticles released from the arrested capsule can repair the damage and thereby enable the capsules to again move along the substrate. Through these studies, we establish guidelines for designing particle-filled microcapsules that perform a ‘repair and go’ function and thus, can be utilized to repair damage in microchannels and microfluidic devices.
Disease control and its selection for damaging plant virus strains in vegetatively propagated staple food crops; a theoretical assessment
Authors: van den Bosch F , Jeger M.J , Gilligan C.A
Published: October 2006

Viral diseases are a key constraint in the production of staple food crops in lesser developed countries. New and improved disease control methods are developed and implemented without consideration of the selective pressure they impose on the virus. In this paper, we analyse the evolution of within-plant virus titre as a response to the implementation of a range of disease control methods. We show that the development of new and improved disease control methods for viral diseases of vegetatively propagated staple food crops ought to take the evolutionary responses of the virus into consideration. Not doing so leads to a risk of failure, which can result in considerable economic losses and increased poverty. Specifically
in vitro
propagation, diagnostics and breeding methods carry a risk of failure due to the selection for virus strains that build up a high within-plant virus titre. For vegetatively propagated crops, sanitation by roguing has a low risk of failure owing to its combination of selecting for low virus titre strains as well as increasing healthy crop density.
Petal herbivory by chrysomelid beetles (Phyllotreta sp.) is detrimental to pollination and seed production in Lepidium papilliferum (Brassicaceae)
Authors: LEAVITT HOLLIE , ROBERTSON IAN C.
Published: October 2006

Abstract 1. Insect herbivory directed at flowers can decrease fruit and seed production by decreasing the attractiveness of a damaged flower to potential pollinators, by disrupting the transfer of pollen between pollinators and stigmas, or both.

2. Effects of petal herbivory by a chrysomelid beetle (Phyllotreta sp.) on pollination and seed production in Lepidium papilliferum (Brassicaceae) were examined.

3. Under natural conditions, flowers with a hole chewed in a petal produced fruit and seed at a significantly lower rate than undamaged flowers (44% vs. 80% respectively). However, when damaged and undamaged flowers were hand pollinated, there was no significant difference in fruit or seed set (84% vs. 80% respectively).

4. Petal herbivory in L. papilliferum disrupts the effectiveness of insect‐mediated pollination, but it does not physically inhibit pollination or seed production.
Proposal of a new staging and grading system of the liver for primary biliary cirrhosis
Authors: Hiramatsu K , Aoyama H , Zen Y , Aishima S , Kitagawa S , Nakanuma Y
Published: October 2006

Aims To define a new histological staging and grading system for primary biliary cirrhosis (PBC), to provide more information reflecting clinical laboratory data and the prognosis to hepatologists.

Methods and results First, 17 histological lesions of PBC were scored in 188 needle liver biopsy specimens. Factor analysis yielded three independent groups of factors: factor 1 (fibrosis, fibrous piecemeal necrosis, orcein‐positive granules, bile plugs, Mallory bodies, feathery degeneration, bile duct loss and atypical ductular proliferation); factor 2 (portal inflammation, eosinophilic infiltration, lymphoid follicles, epithelioid granulomas, interface hepatitis and chronic cholangitis); and factor 3 (interface hepatitis, lobular hepatitis, acidophilic bodies and pigmented macrophages). The eight findings of factor 1, but not factors 2 and 3, were significantly correlated with clinical laboratory data and scores in the Mayo Clinic's prognostic model. Factor 1 lesions may reflect histological progression (staging), while factor 2 and 3 lesions may relate to necroinflammatory activity (grading). Then, we devised a staging and grading system using three lesions (bile duct loss, fibrosis and orcein‐positive granules) from factor 1 and three from factors 2 and 3 (chronic cholangitis, interface hepatitis and lobular hepatitis).

Conclusion This new system might provide more pathological information on PBC patients for hepatologists.
ΔμH+ dependency of in vitro protein translocation into Escherichia coli inner‐membrane vesicles varies with the signal‐sequence core‐region composition
Authors: Nouwen Nico , de Kruijff Ben , Tommassen Jan
Published: October 2006

Signal sequences frequently contain α‐helix‐destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the α‐helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre‐PhoE), a glycine residue at position ‐10 (Gly−10) is thought to be responsible for the break in the α‐helix. We investigated the role of this glycine residue in the translocation process by employing site‐directed mutagenesis. SDS‐PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild‐type PhoE, except when a charged residue (Arg) was substituted for Gly−10. However, the in vitro translocation of all mutant proteins into inverted inner‐membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly−10) was almost independent of the presence of a ΔμH+, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the ΔμH+. Apparently, the ΔμH+ dependency of in vitro protein translocation varies with the signal‐sequence core‐region composition. Furthermore, a proline residue at position ‐10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.
mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae
Authors: Yun Ding‐Fang , Laz Thomas M. , Clements John M. , Sherman Fred
Published: October 2006

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1‐Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild‐type levels. The following major conclusions were reached by measuring β‐galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide‐long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position −3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out‐of‐frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (−3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.
Generation of Campylobacter fetus S‐layer protein diversity utilizes a single promoter on an invertible DNA segment
Authors: Dworkin Joel , Blaser Martin J.
Published: October 2006

Wild‐type strains of Campylobacter fetus contain a monomolecular array of surface layer proteins (SLPs) and vary the antigenicity of the predominant SLP expressed. Reciprocal recombination events among the eight genomic SLP gene cassettes, which encode 97‐ to 149 kDa SLPs, permit this variation. To explore whether SLP expression utilizes a single promoter, we created mutant bacterial strains using insertional mutagenesis by rescue of a marker from plasmids. Experimental analysis of the mutants created clearly indicates that SLP expression solely utilizes the single sapA promoter, and that for variation C. fetus uses a mechanism of DNA rearrangement involving inversion of a 6.2 kb segment of DNA containing this promoter. This DNA inversion positions the sapA promoter immediately upstream of one of two oppositely oriented SLP gene cassettes, leading to its expression. Additionally, a second mechanism of DNA rearrangement occurs to replace at least one of the two SLP gene cassettes bracketing the invertible element. As previously reported promoter inversions in prokaryotes, yeasts and viruses involve alternate expression of at most two structural genes, the ability of C. fetus to use this phenomenon to express one of multiple cassettes is novel.
Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20
Authors: Meijer Wilfried J. J. , Smith Mark , Wake R. Gerry , de Boer Arjo L. , Venema Gerard , Bron Sierd
Published: October 2006

We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated Ter LS20, is the first replication terminator identified on a theta plasmid from a Gram‐positive bacterium. Ter LS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.
The Escherichia coli ribosomal protein S16 is an endonuclease
Authors: Oberto Jacques , Bonnefoy Eliette , Mouray Elisabeth , Pellegrini Olivier , Wikström P. Mikael , Rouvière‐Yaniv Josette
Published: October 2006

The histone‐like protein HU isolated from Escherichia coli exhibited, after several purification steps, a Mg2+‐dependent nuclease activity. We show here that this activity can be dissociated from HU by a denaturation‐renaturation step, and is due to a small fraction of ribosomal protein S16 co‐purifying with HU. S16 is an essential component of the 30S ribosomal particles. We have cloned, overproduced, and purified a histidine‐tagged S16 and shown that this protein is a DNA‐binding protein carrying a Mg2+‐Mn2+‐dependent endonuclease activity. This is an unexpected property for a ribosomal protein.
Tetrapac (tpc), a novel genotype of Neisseria gonorrhoeae affecting epithelial cell invasion, natural transformation competence and cell separation
Authors: Fussenegger Martin , Kahrs Andreas F. , Facius Dirk , Meyer Thomas F.
Published: October 2006

We characterized a novel mutant phenotype (tetrapac, tpc) of Neisseria gonorrhoeae (Ngo) associated with a distinctive rough‐colony morphology and bacterial growth in clusters of four. This phenotype, suggesting a defect in cell division, was isolated from a mutant library of Ngo MS11 generated with the phoA minitransposon TnMax4. The tpc mutant shows a 30% reduction in the overall murein hydrolase activity using Escherichia coli murein as substrate. Tetrapacs can be resolved by co‐cultivation with wild‐type Ngo, indicating that Tpc is a diffusible protein. Interestingly, Tpc is absolutely required for the natural transformation competence of piliated Ngo. Mutants in tpc grow normally, but show a ∼ 10‐fold reduction in their ability to invade human epithelial cells. The tpc sequence reveals an open reading frame of ∼1 kb encoding a protein (Tpc) of 37kDa. The primary gene product exhibits an N‐terminal leader sequence typical of lipoproteins, but palmitoylation of Tpc could not be demonstrated. The ribosomal binding site of tpc is immediately downstream of the translational stop codon of the folC gene coding for an enzyme involved in folic acid biosynthesis and one‐carbon metabolism. The tpc gene is probably co‐transcribed from the folC promoter and a promoter located within the folC gene. The latter promoter sequence shares significant homology with E. coli gearbox consensus promoters. All three mutant phenotypes, i.e. the cell separation defect, the transformation deficiency and the defect in cell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of iap in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus subtilis, all of which are considered to represent murein hydrolase defects.
Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation
Authors: Gentry Daniel R. , Cashel Michael
Published: October 2006

The spoT gene of Escherichia coli encodes a guanosine 3′,5′‐bis(diphosphate) 3′‐pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3′,5′‐bis(diphosphate) synthetase (designated PSII). To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid‐borne deletion mutations for their ability to complement chromosomal mutations defective in each activity. We found that a region containing the first 203 amino acids of the 702‐amino‐acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67–374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein. A ppGppase‐defective Δ1–58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild‐type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation. We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.
Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino‐terminal ATP‐binding domain
Authors: Rajapandi Thavamani , Oliver Donald
Published: October 2006

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP‐binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high‐affinity nucleotide‐binding site, oligonucleotide‐directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine‐specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross‐linking with [α‐32P]‐ATP. This analysis revealed that the nucleotide was cross‐linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP‐binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low‐affinity ATP‐domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high‐affinity ATP‐binding site. These results delimit more precisely the high‐affinity ATP‐binding domain of SecA, indicate the importance of the early amino‐terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de‐insertion from, the inner membrane by the activity of the high‐affinity ATP‐binding domain.
Purification and characterization of a histone‐like protein from the Archaeal isolate AN1, a member of the Thermococcales
Authors: Ronimus Ron S. , Musgrave David R.
Published: October 2006

We have purified and characterized the histone‐like protein, termed HAN1, and an HAN1‐associated DNA‐binding protein (hDBP) from nucleoids of the hyper‐thermophilic Thermococcus‐like AN1. HAN1 is shown to be composed of two subunits, to be thermally stable and to compact DNA in a reversible manner. The N‐terminal sequence of HAN1 shares a high degree of homology with HMf, the histone‐like protein from Methanothermus fervidus. Consistent with this, the toroidal wrapping of DNA by HAN1 resembles that described for HMf. However, significant differences in both twist and writhe components of these complexes are indicated by the 12.0 bp helical repeat produced during hydroxyl radical footprinting with HAN1. Furthermore, the increased stability of HAN1: DNA complexes allows DNA to be protected from thermal denaturation and cleavage by the restriction enzyme TaqI at 65°C. The hDBP, which co‐purified with HAN1, is shown to represent a major portion of the acid‐washed nucleoid protein in AN1 and to enhance the mobility of DNA directly, yet decrease the mobility of HAN1:DNA complexes.
Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species‐specific secretion of proteins via the type II pathway
Authors: Lindeberg Magdalen , Salmond George P. C. , Collmer Alan
Published: October 2006

The type II or Sec‐dependent secretion system is used by diverse Gram‐negative bacteria for secretion of extracellular proteins. Of the 12–15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extracellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species‐specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2006, from E. chrysanthemi EC16, which enables Escherichia coli to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC‐M and outO. We report here the construction of functionally non‐polar mutations in each gene in the outC‐M operon and outS and outB using a polAts strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild‐type copies on pCPP2006. By testing for complementation of each deletion with wild‐type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC‐M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system.
Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose
Authors: Yin Zhikang , Smith Rachel J. , Brown Alistair J. P.
Published: October 2006

The transcription of the yeast FBP1 and PCK1 genes, which encode the gluconeogenic enzymes fructose‐1,6‐bisphosphatase and phosphoenolpyruvate car‐boxykinase, is repressed by glucose. Here, we show that this repression is both very strong and exceptionally sensitive to glucose, being triggered by glucose at concentrations less than 0.005% (0.27 mM). This repression remains operative in yeast mutants carrying any one of the three hexose kinases, but is lost in a triple hxk1, hxk2, glk1 mutant. In addition, 2‐deoxyglucose can trigger the repression, but 6‐deoxy‐glucose cannot, suggesting that internalization and phosphorylation of the glucose is essential for repression to occur. While gluconeogenic gene transcription is subject to the Mig1p‐dependent pathway of glucose repression, the exquisite response to glucose is maintained in hxk2 and mig1 mutants, suggesting that this pathway is not essential for the response. The response can also be triggered by the addition of exogenous cAMP, suggesting that the Ras/cAMP pathway can mediate repression of the FPB1 and PCK1 mRNAs. However, the response is not dependent upon this pathway because it remains intact in Ras, adenyl cyclase and protein kinase A mutants. The data show that yeast cells can detect very low glucose concentrations in the environment, and suggest that several distinct signalling pathways operate to repress FPB1 and PCK1 transcription in the presence of glucose.
RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis
Authors: Prior C. , Tizzani L. , Fukuhara H. , Wésolowski‐Louvel M.
Published: October 2006

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.
Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA
Authors: Stanley Peter , Koronakis Vassilis , Hardie Kim , Hughes Colin
Published: October 2006

The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl‐acyl carrier protein, two independent HlyC recognition domains were identified on the 1024‐residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15–30 amino acids for basal recognition and 50–80 amino acids for wild‐type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAll domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.
Variability of gene order in different Helicobacter pylori strains contributes to genome diversity
Authors: Jiang Qin , Hiratsuka Koji , Taylor Diane E.
Published: October 2006

Considerable genomic microdiversity has been reported previously among Helicobacter pylori isolates. We have constructed genome maps of four unrelated H. pylori strains (NCTC11637, NCTC11639, UA802 and UA861) using pulsed‐field gel electrophoresis (PFGE) with Notl and Nrul, hybridization with extracted PFGE DNA fragments and probing with 17 gene probes. These strains of H. pylori were compared with a fifth unrelated H. pylori strain NCTC11638 mapped previously. Considerable diversity in gene arrangement was evident among the five H, pylori maps, and no consistent gene clustering was found. The association of only four genes, katA (catalase gene), vacA (vacuo‐lating cytotoxin gene), hpaA (a putative adhesin gene), and pfr (bacterial ferritin gene) were generally conserved within approximately the same 25% of the genome; however, the order of these genes also varied. Our study demonstrates that macrodiversity, i.e. variability in gene order, in addition to microdiversity, is a characteristic of the H. pylori genome.
Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production
Authors: Cowley Siobhan C. , Myltseva Svetlana V. , Nano Francis E.
Published: October 2006

Many microbial pathogens, such as Mycobacterium spp. and Salmonella spp., use macrophage intracellular growth or antigenic variation as mechanisms for avoiding the host immune system. In this work we present evidence to show that the intracellular pathogen Francisella tularensis uses phase variation to alter antigenicity and the host macrophage nitric oxide response simultaneously, thereby modulating its intracellular growth. The lipopolysaccharide (LPS) and lipid A of F. tularensis fails to stimulate production of significant levels of nitric oxide (NO) by rat macrophages. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing microbial intramacrophage growth. Similarly, lipid A isolated from these variants stimulates increased levels of NO production. A reverse phase shift can occur, which returns the LPS to the original antigenic form, reduces NO production, and restores intramacrophage growth. These findings represent the first demonstration of a phase‐variation phenomenon which modulates intracellular growth and an innate immune response. Furthermore, these results suggest that a microbial pathogen can exploit macrophage NO production for its own benefit, perhaps by prolonging the host‐pathogen association during the acute phase of disease or during the process of establishing a carrier state.
Generation of unmarked directed mutations in mycobacteria, using sucrose counter‐selectable suicide vectors
Authors: Pelicic Vladimir , Reyrat Jean‐Marc , Gicquel Brigitte
Published: October 2006

The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene‐replacement events into Mycobacterium smegmatis. A sucrose counter‐selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrFKm) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one‐step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic‐exchange mutants. Two‐step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.
Protein secretion by heterologous bacterial ABC‐transporters: the C‐terminus secretion signal of the secreted protein confers high recognition specificity
Authors: Duong F. , Lazdunski A. , Murgier M.
Published: October 2006

Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) is released by its own specific secretion machinery which is an ABC‐transporter. Despite sequence similarities between components of ABC‐transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC‐transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C‐terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated a‐haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the trans‐locator which confers specificity to the secretion process. Secretion was size‐dependent. However inclusion of glycine‐rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20kDa larger than the normal maximal size.
Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae
Authors: Alioing Geneviève , Granadel Chantal , Morrison Donald A. , Claverys Jean‐Pierre
Published: October 2006

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide‐binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild‐type strain and we investigated the response of the various mutants to treatment with synthetic competence‐stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide‐binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide‐binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two‐component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid‐insertion mutation abolished competence completely. In addition, the triple AmiA‐AliA‐AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.
The Bacillus subtilis soj‐spo0J locus is required for a centromere‐like function involved in prespore chromosome partitioning
Authors: Sharpe Michaela E. , Errington Jeffery
Published: October 2006

During sporulation in Bacillus subtilis a small prespore cell is formed by an asymmetric cell division. Pre‐spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement. We show that the spo0J gene is needed to specify the orientation of the chromosome at the time of polar division and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum. Both phenotypes may arise by disruption of a centromere‐like apparatus that anchors the oriC region of the prespore chromosome in the pole of the cell.
CheC and CheD interact to regulate methylation of Bacillus subtilis methyl‐accepting chemotaxis proteins
Authors: Rosario Mia Mae L. , Ordal George W.
Published: October 2006

In this study, we have demonstrated that two unique proteins inBacillus subtilischemotaxis, CheC and CheD, interact. We have shown this interaction both by using the yeast two‐hybrid system and by precipitation ofin vitrotranslated products using glutathione‐S‐transferase fusions and glutathione agarose beads. We have also shown that CheC inhibitsB. subtilisCheR‐mediated methylation ofB. subtilismethyl‐accepting chemotaxis proteins (MCPs) but not ofEscherichia coliMCPs. It was previously reported thatcheCmutants tend to swim smoothly and do not adapt to addition of attractant;cheDmutants have very poorly methylated MCPs and are very tumbiy, similar tocheAmutants. We hypothesize that CheC exerts its effect on MCP methylation inB. subtilisby controlling the binding of CheD to the MCPs. In absence of CheD, the MCPs are poor substrates for CheR and appear to tie up, rather than activate, CheA. The regulation of CheD by CheC may be part of a unique adaptation system for chemotaxis inB. subtilis, whereby high levels of CheY‐P brought about by attractant addition would allow CheC to interact with CheD and consequently leave the MCPs, reducing CheA activity and hence the levels of CheY‐P.