bluuebunny/crossref_metadata_2025 huggingface dataset:
Classification of 3T1R Parallel Manipulators Based on Their Wrench Graph
Authors: Amine Semaan , Mokhiamar Ossama , Caro Stéphane
Published: November 2016

This paper presents a classification of 3T1R parallel manipulators (PMs) based on the wrench graph. By using the theory of reciprocal screws, the properties of the three-dimensional projective space, the wrench graph, and the superbracket decomposition of Grassmann–Cayley algebra, six typical wrench graphs for 3T1R parallel manipulators are obtained along with their singularity conditions. Furthermore, this paper shows a way in which each of the obtained typical wrench graphs can be used in order to synthesize new 3T1R parallel manipulator architectures with known singularity conditions and with an understanding of their geometrical properties and assembly conditions.
Numerical Simulation of Noninteger Order System in Subdiffusive, Diffusive, and Superdiffusive Scenarios
Authors: Owolabi Kolade M. , Atangana Abdon
Published: November 2016

In this work, we investigate both the mathematical and numerical studies of the fractional reaction–diffusion system consisting of spatial interactions of three components’ species. Our main result is based on the analysis of the model for linear stability. Mathematical analysis of the main equation shows that the dynamical system is both locally and globally asymptotically stable. We further propose a theorem which guarantees the existence and permanence of the three species. We formulate a viable numerical methods in space and time. By adopting the Fourier spectral approach to discretize in space, the issue of stiffness associated with the fractional-order spatial derivatives in such system is removed. The resulting system of ordinary differential equations (ODEs) is advanced with the exponential time-differencing method of ADAMS-type. The complexity of the dynamics in the system which we discussed theoretically are numerically presented through some numerical simulations in 1D, 2D, and 3D to address the points and queries that may naturally arise.
Numerical Study in a Mini Trochoidal-Gear Pump With Multi-Meshing Contact Points
Authors: Gamez-Montero P. J. , Castilla R. , Buza A. , Khamashta M. , Codina E.
Published: November 2016

A numerical study in a mini trochoidal-gear pump based on an ingenious strategy to adapt the background mesh to the profiles shape and a new boundary condition is presented. The strategy achieves a final background mesh better adapted to the geometries and, additionally, the cells are more homogenous and less distorted when the trochodial-profiles are moved. The new boundary condition to simulate the multi-meshing contact points is applied at every time step by means of the viscous wall approach, a home-made ad-hoc code, which has been integrated in an OpenFOAM solver that also includes the dynamic mesh functionality by deforming the mesh following the trochoidal-gear rotation.

The presented numerical study establishes the basis that will allow simulating more realistic aspects of a new design of a mini trochoidal-gear pump once the need for a continuous fluid domain is overcome reproducing actual contacts between the rotors and will guide to a better understanding of the new design.
Investigation of Steady-State Flow Forces in Spool Valves of Different Geometries and at Different Oil Temperatures With the Help of Measurements and CFD Simulations
Authors: Bordovsky Patrik , Murrenhoff Hubertus
Published: November 2016

When designing an actuator for a spool type directional control valve, axial forces acting on the spool have to be estimated. The steady-state flow force is the dominant axial force, which usually acts in the closing direction of the valve. However, many factors such as the valve geometry and the oil properties influence the flow force characteristics. Investigations regarding their effects on steady-state flow forces are described within this paper.

Different spool geometries of a test 2/2-way spool valve are used for steady-state flow force measurements at different oil temperatures. The measurement data are used for validation of CFD simulations, which are carried out to scrutinise the flow inside the valve. Besides the steady-state flow forces, the fluid flow angles at the inlet and the outlet of the spool chamber are analysed.

The results show that the spool geometry has a significant influence both on the flow rate and the steady-state flow force characteristics. Especially, the shape of the control edge has an impact on the flow patterns and on the magnitudes of steady-state flow forces. Moreover, the inlet and outlet fluid flow angles do not correlate with the expected values, which are commonly used for an analytical estimation of the flow forces. Furthermore, the oil temperature leads to quantitative deviations of the steady-state flow forces.
Reducing Friction and Wear in Common-Rail Pumps by Grooved and Contoured Pistons
Authors: Rückert Marcel , Heitzig Stefan , Murrenhoff Hubertus
Published: November 2016

Within the cluster of excellence “Tailor-made Fuels from Biomass” at RWTH Aachen University new biofuels are developed and investigated. Because common-rail injection pumps are generally lubricated by the fuel itself, the tribological characteristics of the fuel candidates is of interest. The lubricity and viscosity of the alternative fuels differ from diesel. Hence, a reliable function of the tribological contacts, which are designed for the operation with diesel, cannot be guaranteed. To achieve a reliable operation even for fuels with, for instance, a lower viscosity or worse lubricity an optimisation of the tribological contacts is necessary.

The focus of the investigations presented in this paper lies on the piston-cylinder-contact. Prior to the simulative study the losses in a pump operating with various fuel candidates are quantified by means of efficiency measurements. By these measurements the great impact of the fuel’s rheology on the pump performance is clarified.

Based on detailed EHD-simulations with various fuels under typical operating conditions, an optimisation of the piston-cylinder-contact is presented. The optimisation aims for the reduction of solid friction by changing the pressure field on the piston. The approaches can basically be separated into grooved and contoured pistons. The potential of the different approaches is discussed based on simulation results and effects, which occur in the lubricating film of the optimised contacts.
Hardware-in-the-Loop (HIL) Simulation of a Quarter Vehicle Model Using a Multi-Axis Simulation Table (MAST)
Authors: Alfadhli Abdulaziz , Darling Jocelyn , Hillis Andrew J.
Published: November 2016

The reliable and repeatable experimental testing of automotive components is a challenge, especially when human occupants are involved. In most circumstances full vehicle testing over a range of road conditions is used even though this is expensive, difficult and time-consuming. In this work an experimental platform that compensates for the vehicle motion using the principle of hardware-in-the-loop (HIL) simulation is developed using a multi-axis simulation table (MAST) available in the Center for Power Transmission and Motion Control (PTMC) laboratory at the University of Bath. The MAST was tested in both the time and frequency domains with a range of road profiles including a random and single bump in the low frequency range (0.5–25 Hz). Also, uncertainties in the vehicle suspension characteristics such as the damping coefficient, the stiffness rate and the vehicle mass are examined. The experimental results in the frequency and time domain show that the MAST can be used to reproduce the dynamic characteristics of a quarter vehicle model with an excellent degree of agreement with the simulated response.
Improved Exact Solution for Axial FSI Model of Liquid-Pipes Considering Complex Constraints
Authors: Xu Yuanzhi , Jiao Zongxia , Shang Yaoxing , Wu Shuai
Published: November 2016

The fluid-structure interaction (FSI) can be observed significantly in the axial vibration of the liquid-filled pipe system. When distributed friction was neglected, the model could be solved with an exact solution without numerical error, developed from the method of characteristics (MOC). Then time-line interpolations have been employed to reduce the high time cost and retain the accuracy, named as the improved exact solution. For the purpose of practical applications, series connection of double-pipes is discussed in this paper. And complex constraints, including elastic, damping and inertial effects at connections, are studied. Models and methods are validated by numerical cases.
Effects of interface states and near interface traps on the threshold voltage stability of GaN and SiC transistors employing SiO2 as gate dielectric
Authors: Fiorenza Patrick , Greco Giuseppe , Giannazzo Filippo , Iucolano Ferdinando , Roccaforte Fabrizio
Published: November 2016

This paper reports on the effects of interface states and near interface traps on the behavior of GaN and SiC transistors employing SiO2 as gate dielectric, emphasizing the role of these interfacial charged traps on the threshold voltage stability of the devices. Capacitance, conductance, and current measurements, carried out as a function of the frequency, were used to characterize the GaN- and SiC-metal-oxide-semiconductor systems. In these systems, although postoxide deposition annealing treatments reduce the interface states density, the presence of near interface traps can induce an anomalous behavior of the current conduction, accompanied by a threshold voltage instability. The transfer characteristics of the transistors acquired in an appropriate bias range enabled to quantify the density of these traps in the order of 1011 cm−2.
An Enhanced Emtricitabine-Loaded Long-Acting Nanoformulation for Prevention or Treatment of HIV Infection
Authors: Mandal Subhra , Belshan Michael , Holec Ashley , Zhou You , Destache Christopher J.
Published: November 2016

Among various FDA-approved combination antiretroviral drugs (cARVs), emtricitabine (FTC) has been a very effective nucleoside reverse transcriptase inhibitor. Thus far, FTC is the only deoxycytidine nucleoside analog. However, a major drawback of FTC is its large volume distribution (averaging 1.4 liters/kg) and short plasma half-life (8 to 10 h), necessitating a high daily dosage. Thus, we propose an innovative fabrication method of loading FTC in poly(lactic-co-glycolic acid) polymeric nanoparticles (FTC-NPs), potentially overcoming these drawbacks. Our nanoformulation demonstrated enhanced FTC loading (size of <200 nm and surface charge of −23 mV) and no to low cytotoxicity with improved biocompatibility compared to those with FTC solution. An
ex vivo
endosomal release assay illustrated that NP entrapment prolongs FTC release over a month. Intracellular retention studies demonstrate sustained FTC retention over time, with approximately 8% (24 h) to 68% (96 h) release with a mean retention of ∼0.74 μg of FTC/10
5
cells after 4 days. An
in vitro
HIV-1 inhibition study demonstrated that FTC-NP treatment results in a 50% inhibitory concentration (IC
50
) ∼43 times lower in TZM-bl cells (0.00043 μg/ml) and ∼3.7 times lower (0.009 μg/ml) in peripheral blood mononuclear cells (PBMCs) than with FTC solution (TZM-bl cells, 0.01861, and PBMCs, 0.033 μg/ml). Further, on primary PBMCs, FTC-NPs also illustrate an HIV-1 infection blocking efficacy comparable to that of FTC solution. All the above-described studies substantiate that FTC nanoformulation prolongs intracellular FTC concentration and inhibition of HIV infection. Therefore, FTC-NPs potentially could be a long-acting, stable formulation to ensure once-biweekly dosing to prevent or treat HIV infection.
Penicillin-Binding Protein 3 Is Essential for Growth of Pseudomonas aeruginosa
Authors: Chen Wei , Zhang Yong-Mei , Davies Christopher
Published: November 2016

Penicillin-binding proteins (PBPs) function as transpeptidases, carboxypeptidases, or endopeptidases during peptidoglycan synthesis in bacteria. As the well-known drug targets for β-lactam antibiotics, the physiological functions of PBPs and whether they are essential for growth are of significant interest. The pathogen
Pseudomonas aeruginosa
poses a particular risk to immunocompromised and cystic fibrosis patients, and infections caused by this pathogen are difficult to treat due to antibiotic resistance. To identify potential drug targets among the PBPs in
P. aeruginosa
, we performed gene knockouts of all the high-molecular-mass (HMM) PBPs and determined the impacts on cell growth and morphology, susceptibility to β-lactams, peptidoglycan structure, virulence, and pathogenicity. Disruptions of the transpeptidase domains of most HMM PBPs, including double disruptions, had only minimal effects on cell growth. The exception was PBP3, where cell growth occurred only when the protein was conditionally expressed on an integrated plasmid. Conditional deletion of PBP3 also caused a defect in cell division and increased susceptibility to β-lactams. Knockout of PBP1a led to impaired motility, and this observation, together with its localization at the cell poles, suggests its involvement in flagellar function. Overall, these findings reveal that PBP3 represents the most promising target for drug discovery against
P. aeruginosa
, whereas other HMM PBPs have less potential.
Pharmacokinetics, Distribution, Metabolism, and Excretion of Omadacycline following a Single Intravenous or Oral Dose of
14
C-Omadacycline in Rats
Authors: Lin Wen , Flarakos Jimmy , Du Yancy , Hu Wenyu , He Handan , Mangold James , Tanaka S. Ken , Villano Stephen
Published: November 2016

The absorption, distribution, metabolism, and excretion (ADME) of omadacycline, a first-in-class aminomethylcycline antibiotic with a broad spectrum of activity against Gram-positive, Gram-negative, anaerobic, and atypical bacteria, were evaluated in rats. Tissue distribution was investigated by quantitative whole-body autoradiography in male Long-Evans Hooded (LEH) rats. Following an intravenous (i.v.) dose of 5 mg/kg of body weight, radioactivity widely and rapidly distributed into most tissues. The highest tissue-to-blood concentration ratios (t/b) were observed in bone mineral, thyroid gland, and Harderian gland at 24 h post-i.v. dose. There was no evidence of stable accumulation in uveal tract tissue, suggesting the absence of a stable binding interaction with melanin. Following a 90 mg/kg oral dose in LEH rats, the highest t/b were observed in bone mineral, Harderian gland, liver, spleen, and salivary gland. The plasma protein binding levels were 26% in the rat and 15% to 21% in other species. Omadacycline plasma clearance was 1.2 liters/h/kg, and its half-life was 4.6 h; the steady-state volume of distribution (Vss) was 6.89 liters/kg. Major circulating components in plasma were intact omadacycline and its epimer. Consistent with observations in human, approximately 80% of the dose was excreted into the feces as unchanged omadacycline after i.v. administration. Fecal excretion was primarily the result of biliary excretion (∼40%) and direct gastrointestinal secretion (∼30%). However, urinary excretion (∼30%) was equally prominent after i.v. dosing.
Altered Plasmodium falciparum Sensitivity to the Antiretroviral Protease Inhibitor Lopinavir Associated with Polymorphisms in
pfmdr1
Authors: Sonoiki Ebere , Nsanzabana Christian , Legac Jennifer , Sindhe Kirthana M. V. , DeRisi Joseph , Rosenthal Philip J.
Published: November 2016

The HIV protease inhibitor lopinavir inhibits
Plasmodium falciparum
aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected
in vitro
over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in
pfmdr1
and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.
Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance
Authors: Montealegre Maria Camila , Roh Jung Hyeob , Rae Meredith , Davlieva Milya G. , Singh Kavindra V. , Shamoo Yousif , Murray Barbara E.
Published: November 2016

Ampicillin resistance in
Enterococcus faecium
is a serious concern worldwide, complicating the treatment of
E. faecium
infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in
E. faecium
. The three known
E. faecium
clades showed sequence variations in the
pbp5
gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among
E. faecium
clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the
E. faecium
clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in
E. faecium
. We studied
pbp5
mRNA levels and PBP5 protein levels as well as the genetic environment upstream of
pbp5
in 16
E. faecium
strains that belong to the different
E. faecium
clades and for which the ampicillin MICs covered a wide range. Our results found that
pbp5
and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of
pbp5
, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the
E. faecium
clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of
pbp5
as likely contributors to the differential expression of ampicillin resistance.
Listeria monocytogenes-Induced Cell Death Inhibits the Generation of Cell-Mediated Immunity
Authors: Theisen Erin , Sauer John-Demian
Published: November 2016

The influence of cell death on adaptive immunity has been studied for decades. Despite these efforts, the intricacies of how various cell death pathways shape immune responses in the context of infection remain unclear, particularly with regard to more recently discovered pathways such as pyroptosis. The emergence ofListeria monocytogenesas a promising immunotherapeutic platform demands a thorough understanding of how cell death induced in the context of infection influences the generation of CD8+T-cell-mediated immune responses. To begin to address this question, we designed strains ofL. monocytogenesthat robustly activate necrosis, apoptosis, or pyroptosis. We hypothesized that proinflammatory cell death such as necrosis would be proimmunogenic while apoptosis would be detrimental, as has previously been reported in the context of sterile cell death. Surprisingly, we found that the activation of any host cell death in the context ofL. monocytogenesinfection inhibited the generation of protective immunity and specifically the activation of antigen-specific CD8+T cells. Importantly, the mechanism of attenuation was unique for each type of cell death, ranging from deficits in costimulation in the context of necrosis to a suboptimal inflammatory milieu in the case of pyroptosis. Our results suggest that cell death in the context of infection is different from sterile-environment-induced cell death and that inhibition of cell death or its downstream consequences is necessary for developing effective cell-mediated immune responses usingL. monocytogenes-based immunotherapeutic platforms.
Activation of a cryptic gene by excision of a DNA fragment
Authors: Parker L L , Betts P W , Hall B G
Published: November 2016

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.
Structure and transcription of the allantoate permease gene (DAL5) from Saccharomyces cerevisiae
Authors: Rai R , Genbauffe F S , Cooper T G
Published: November 2016

We determined the nucleotide sequence of the DAL5 gene, which encodes a component of the allantoate transport system. Translation of the sequence revealed that the DAL5 gene product is highly hydrophobic. It possesses an alternating motif of hydrophilic sequences that can potentially be folded into alpha-helices and hydrophobic sequences that can potentially be folded into beta-pleated sheets. These are expected characteristics of an integral membrane protein, which correlate well with DAL5 gene function. S1 protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range. This pattern of fragments was not affected by growth conditions of the cells or the conditions of the assay.
Drug-induced relaxation of supercoiled plasmid DNA in Bacillus subtilis and induction of the SOS response
Authors: Osburne M S , Zavodny S M , Peterson G A
Published: November 2016

Whereas treatment with many different drugs led to induction of the SOS response in Bacillus subtilis, only inhibitors of DNA gyrase subunit B and, unexpectedly, polyether antibiotics (membrane ionophores) led to relaxation of supercoiled plasmid DNA. However, treatment with DNA gyrase subunit B inhibitors but not with polyethers led to SOS induction. Thus, the presence of underwound supercoiled DNA was not sufficient to induce the SOS response. Possible mechanisms by which polyethers induce relaxation of supercoiled DNA in vivo are discussed.
Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides
Authors: Falcone D L , Quivey R G , Tabita F R
Published: November 2016

Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme.
Cyclic AMP and cell division in Escherichia coli
Authors: D'Ari R , Jaffé A , Bouloc P , Robin A
Published: November 2016

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.
Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches
Authors: Lee C C , Kohara Y , Akiyama K , Smith C L , Craigen W J , Caskey C T
Published: November 2016

The termination of protein synthesis in Escherichia coli requires two codon-specific factors termed RF1 and RF2. RF1 mediates UAA- and UAG-directed termination, while RF2 mediates UAA- and UGA-directed termination. The genes encoding these factors have been isolated and sequenced, and RF2 was found to be encoded in two separate reading frames. The map position of RF1 has been reported as 27 min on the E. coli chromosome, while the RF2 map position has not yet been identified. In this study, two new and independent methods for gene mapping, using pulsed field gel electrophoresis and an ordered bacteriophage library spanning the entire chromosome, were used to localize the map position of the RF2 gene. In addition, the location of the RF1 gene was more precisely defined. The RF2 gene is located at 62.3 min on the chromosome, while the RF1 gene is located at 26.7 min. This approach to mapping cloned genes promises to be a rapid and simple means for determining the gene order of the genome.
Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death
Authors: Suit J L , Luria S E
Published: November 2016

Expression of the kil gene of the ColE1 plasmid in certain classes of Escherichia coli mutants (Kilr) resistant to kil-caused cell death brought about release of periplasmic enzymes and of colicin. Phospholipase A was present but was not activated by kil expression in any of the mutants. This indicates that in these mutants the various effects of kil gene expression have become dissociated.
Cloning and expression of genes encoding pheromone-inducible antigens of Enterococcus (Streptococcus) faecalis
Authors: Christie P J , Kao S M , Adsit J C , Dunny G M
Published: November 2016

Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the Tra150 and Tra130 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing Tn5 or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tra130 and Tra150.
Molecular cloning, sequencing, and mapping of the bacteriophage T2 dam gene
Authors: Miner Z , Hattman S
Published: November 2016

Bacteriophage T2 codes for a DNA-(adenine-N6)methyltransferase (Dam), which is able to methylate both cytosine- and hydroxymethylcytosine-containing DNAs to a greater extent than the corresponding methyltransferase encoded by bacteriophage T4. We have cloned and sequenced the T2 dam gene and compared it with the T4 dam gene. In the Dam coding region, there are 22 nucleotide differences, 4 of which result in three coding differences (2 are in the same codon). Two of the amino acid alterations are located in a region of homology that is shared by T2 and T4 Dam, Escherichia coli Dam, and the modification enzyme of Streptococcus pneumoniae, all of which methylate the sequence 5' GATC 3'. The T2 dam and T4 dam promoters are not identical and appear to have slightly different efficiencies; when fused to the E. coli lacZ gene, the T4 promoter produces about twofold more beta-galactosidase activity than does the T2 promoter. In our first attempt to isolate T2 dam, a truncated gene was cloned on a 1.67-kilobase XbaI fragment. This construct produces a chimeric protein composed of the first 163 amino acids of T2 Dam followed by 83 amino acids coded by the pUC18 vector. Surprisingly, the chimera has Dam activity, but only on cytosine-containing DNA. Genetic and physical analyses place the T2 dam gene at the same respective map location as the T4 dam gene. However, relative to T4, T2 contains an insertion of 536 base pairs 5' to the dam gene. Southern blot hybridization and computer analysis failed to reveal any homology between this insert and either T4 or E. coli DNA.
Molecular cloning of genes that specify virulence in Pseudomonas solanacearum
Authors: Xu P L , Leong S , Sequeira L
Published: November 2016

The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.
Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins
Authors: Ward E S , Ellar D J
Published: November 2016

Two homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985).
Regulation of tryptophan biosynthesis in Caulobacter crescentus
Authors: Ross C M , Winkler M E
Published: November 2016

We present an analysis of the expression of the trpE gene and the trpFBA operon in the dimorphic bacterium Caulobacter crescentus. The catalytic activity of component I of anthranilate synthase, the product of the trpE gene, was efficiently inhibited by tryptophan, the end product of the pathway, which suggests that tryptophan biosynthesis is likely controlled at the pathway level in C. crescentus. However, trpFBA mRNA levels and trpE enzyme levels did not vary significantly in wild-type C. crescentus in response to the presence of tryptophan in the growth medium or to growth in minimal versus rich medium. This lack of regulation of the trpE, trpF, trpB, and trpA genes is consistent with the idea that oligotrophic bacteria, such as C. crescentus, do not utilize regulatory mechanisms that greatly alter the biosynthetic capabilities in exponentially growing cells. In contrast, mRNA levels from the 5'-untranslated region and the upstream gene (usg) coding region increased dramatically in C. crescentus trpD or hisB auxotrophs starved for tryptophan or histidine, respectively. Surprisingly, concomitant increases in mRNA levels were not detected from the trpF, trpB, or trpA coding regions downstream in the operon. Thus, severe starvation of C. crescentus for amino acids appears to elicit a strong, general transcriptional response that is not observed in bacteria growing exponentially in medium lacking amino acids.
Cloning and physical mapping of RNA polymerase genes from Methanobacterium thermoautotrophicum and comparison of homologies and gene orders with those of RNA polymerase genes from other methanogenic archaebacteria
Authors: Schallenberg J , Moes M , Truss M , Reiser W , Thomm M , Stetter K O , Klein A
Published: November 2016

The structural genes encoding the four largest subunits of RNA polymerase, A, B', B", and C, were physically mapped in Methanobacterium thermoautotrophicum Winter. The genes formed a cluster in the order B", B', A, C and had a common orientation. DNA hybridization experiments yielded different degrees of homology between RNA polymerase gene sequences of different species of Methanobacterium and Methanococcus voltae. No homology was detectable between Methanobacterium thermoautotrophicum and Methanosarcina barkeri. From Southern hybridization experiments in which probes of the four genes from Methanobacterium thermoautotrophicum Winter and restriction digests of the genomic DNAs of the different methanogens were used, a common gene order of the RNA polymerase genes could be deduced.
Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43
Authors: McHenney M A , Baltz R H
Published: November 2016

A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.
Isolation and biochemical characterization of the S-layer protein from a pathogenic Aeromonas hydrophila strain
Authors: Dooley J S , McCubbin W D , Kay C M , Trust T J
Published: November 2016

The regular surface protein array (S layer) present on Aeromonas hydrophila TF7 is composed of a single species of protein of apparent molecular weight 52,000. This protein was extracted from whole cells by treatment with 0.2 M glycine hydrochloride (pH 3.0). The protein was purified to homogeneity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. Amino acid composition analysis showed that the protein contained 520 residues per molecule, 41% of which were hydrophobic. Cysteine was absent. A pI of 4.6 was determined for the protein, and only a single isoelectric form was detected. The purified protein displayed the hydrophobic characteristic of binding to octyl-Sepharose gels, but the salt aggregation test showed that it did not confer hydrophobicity to the cell surface when present as an intact S layer. The molecule aggregated strongly in aqueous solution as determined by sedimentation equilibrium studies. Circular dichroism spectra showed that the S-layer protein was composed of a large amount of beta-sheet (approximately 44%), a limited amount of alpha-helix (19%), and 12% beta-turn, with the remainder of the molecule being aperiodic. No significant difference in secondary structure content was measured in the presence of the metal chelator EDTA. The N-terminal amino acid sequence was determined for the first 30 residues. No sequence homology with other S-layer proteins was found.
Genetic mapping and physiological consequences of metE mutations of Bacillus subtilis
Authors: Wabiko H , Ochi K , Nguyen D M , Allen E R , Freese E
Published: November 2016

Three metE mutations of Bacillus subtilis, which cause cells to have a 25- to 200-fold decrease in L-methionine S-adenosyltransferase (EC 2.5.1.6) activity, were mapped between bioB and thr. The corresponding three metE mutants contained three- to fourfold less intracellular S-adenosylmethionine (SAM) but at least sevenfold more methionine than the metE+ strain when grown in synthetic medium. This indicates a strong feedback control of SAM on its synthesis. However, only the metE2 strain, with the lowest SAM concentration, grew at a slightly lower rate than the parent, which showed that an intracellular concentration of about 25 microM SAM was critical for growth at the normal rate. Neither DNA methylation (measured by bacteriophage luminal diameter 105 restriction) nor sporulation was affected at this low SAM concentration. Addition of methionine to the growth medium caused an increase in the pool of SAM in some but not all metE mutants. Coaddition of adenine did not change this result. However, the extent of sporulation (induced by mycophenolic acid) was decreased 50-fold in all mutants by the addition of methionine and adenine. Therefore, the combination of methionine and adenine suppresses sporulation regardless of whether it causes an increase in the level of SAM.
Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli
Authors: Giaever H M , Styrvold O B , Kaasen I , Strøm A R
Published: November 2016

It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.
Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement
Authors: Howell E E , Foster P G , Foster L M
Published: November 2016

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.
Molecular cloning and characterization of supQ/newD, a gene substitution system for the leuD gene of Salmonella typhimurium
Authors: Stover C K , Kemper J , Marsh R C
Published: November 2016

The isopropylmalate isomerase of Salmonella typhimurium and Escherichia coli is a complex of the leuC and leuD gene products. The supQ/new D gene substitution system in S. typhimurium restores leucine prototrophy to leuD mutants of S. typhimurium. Previous genetic evidence supports a model that indicates the replacement of the missing LeuD polypeptide by the newD gene product. This model proposed that this gene substitution is possible when a mutation at the supQ locus (near newD) liberates unaltered newD polypeptide from its normal complex with the supQ protein product. In this study, recombinant plasmids carrying newD, supQ, or both were transformed into E. coli and S. typhimurium strains deleted for the leuD and supQ genes to test the supQ/newD gene substitution model for suppression of leucine auxotrophy. It was determined that the newD gene encodes a 22-kilodalton polypeptide which can restore leucine prototrophy to leuD deletion strains and that a functional supQ gene prevents this suppression. It was also determined that the supQ and newD genes are separated by a gene encoding a 50-kilodalton protein, pB. While there is extensive DNA sequence homology between the leucine operons of S. typhimurium and E. coli, DNA hybridization experiments did not indicate substantial homology between the newD and leuD genes. These data, taken together with previously obtained genetic data, eliminate the possibility that supQ and newD are recently translocated segments of the leucine operon.
Minimal region necessary for autonomous replication of pTAR
Authors: Gallie D R , Kado C I
Published: November 2016

The native 44-kilobase-pair plasmid pTAR, discovered in a grapevine strain of Agrobacterium tumefaciens, contains a single origin of DNA replication confined to a 1.0-kilobase-pair region of the macromolecule. This region (ori) confers functions sufficient for replication in Agrobacterium and Rhizobium species but not in Pseudomonas solanacearum, Pseudomonas glumae, Pseudomonas syringae pv. savastanoi, Xanthomonas campestris pv. campestris, and Escherichia coli. ori contains a repA gene that encodes a 28,000-dalton protein required for replication. Nucleotide sequencing of repA and its promoter region revealed four 8-base-pair palindromic repeats upstream of the repA coding region. Deletion of these repeats alters repA expression and plasmid copy number. Downstream of repA are three additional repeats in a region essential for replication. A locus responsible for plasmid partitioning (parA) and a putative second locus regulating plasmid copy number are part of the origin region and are required for stable plasmid maintenance.
Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae
Authors: Drebot M A , Barnes C A , Singer R A , Johnston G C
Published: November 2016

Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties. The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth. We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway. We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase. The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.