bluuebunny/crossref_metadata_2025 huggingface dataset:
Improvement of Pipeline Fatigue Life Estimation
Authors: Tiku Sanjay , Dinovitzer Aaron , Semiga Vlad , Piazza Mark , Jones Tom
Published: November 2016

Fracture mechanics methodologies for calculating fatigue lives have been successfully applied by pipeline operators to estimate integrity reassessment intervals. Their application in the definition of pipeline system fatigue lives has been overly conservative in actual practice. The source and magnitude of the conservatism inherent in the calculated fatigue life estimates needs to be identified so operators have a better indicator of when reassessments should take place. The pipe life estimation is especially critical for Electric Resistance Weld (ERW) and Electric Flash Weld (EFW) pipeline systems with longitudinally oriented defects.

Prior work on improving fatigue life was initiated through studies completed by Pipeline Research Council International, Inc. (PRCI) to evaluate the sources of differences between fatigue life estimates produced by industry fatigue analysis software and different metallurgists. Two significant sources of conservatism in the fatigue life estimation process were identified: the fatigue crack growth rate (da/dN) and the bulging correction factor applied to axial surface flaws. The experimental and numerical simulation techniques considering the impact of these factors on rate of fatigue crack growth of pipeline axially oriented defects are described in this paper.

Finite element modeling was used to simulate pipe bulging in the presence of axial flaws. The effect of the pipe thickness, diameter and flaw geometry was compared with treatments included in existing defect assessment standards. The results illustrate that for longer and deeper flaws existing treatments over represent the local bending due to pipe wall bulging. This results in unnecessarily conservative (shorter) fatigue life estimates.

The crack growth rate (da/dN) was measured in a compact tension specimen material fatigue testing program. The test results included a range of ERW and EFW pipe materials with varying vintages and grades. The measured fatigue crack growth rate for the materials tested was found to be lower than that recommended by existing industry standards. This adds to the over conservatism of current approaches.

The numerical simulation and materials testing results and related recommendations presented in this paper are compared to existing codified treatments to quantify the level of conservatism inherent in the current state of practice. Recommendations are provided to enhance the precision and better manage conservatism in fatigue crack growth rate calculations. Increased accuracy serves to improve integrity management and would be of interest to pipeline operators, consultants and regulators.
Entrapped Gas and Vapor Formation Influence on Surge Pressure in Liquid Pipeline Systems
Authors: Cheng David , Seng Borith
Published: November 2016

Predicting the effects of entrapped gas or vapor formation on surge is very important in design and operation of liquid pipelines. This paper identified the scenarios in which entrapped air and vapor formation need to be considered in pipeline operation and design. Useful modeling methods utilizing common liquid pipeline transient hydraulics software are provided. Validation of the presented methods was completed using experimental data from published literature. Examples are presented in showing the implementation of the provided modeling methods on real pipeline design scenarios. Finally, advantages and limitations of the presented methods was discussed. The methods presented in this paper enable pipeline operators and design engineers to properly estimate the complicated surge issues such as the influence of air bubble venting and column separation and collapse using commonly available single phase hydraulics tools. The operators and engineers will benefit from the provided methods in finding and validating reliable surge mitigation solutions and creating pipeline design with higher integrity level. The paper also presents the limitation of the methods and continuous improvements that can be achieved in the future.
Effect of Cold-Wire Addition in the TSAW Process on Microstructure and Mechanical Properties of the HAZ of X70 Microalloyed Pipeline Steel
Authors: Mohammadijoo Mohsen , Kenny Stephen , Collins Laurie , Henein Hani , Ivey Douglas G.
Published: November 2016

Microalloyed steels can achieve a good combination of strength and toughness through appropriate alloy design and thermomechanical controlled processing (TMCP). However, the mechanical properties can deteriorate as a result of the high heat input and thermal cycles that the steel experiences during welding. It is generally accepted that the portion of the heat affected zone (HAZ) adjacent to the fusion line, i.e., the coarse grain heat affected zone (CGHAZ), which is characterized by coarse grains and martensite-austenite (M-A) constituents, is the region with poorer toughness relative to the rest of the steel. In the present research work, modification to the conventional tandem submerged arc welding (TSAW) process is carried out by the addition of a cold wire during welding (CWTSAW), which induces changes to the geometry and properties of the weld joint. Microstructural analysis, mechanical property investigation and geometry analysis indicate overall improvement in the weld and the HAZ properties after cold wire addition. These improvements are explained in terms of an increase in the deposition rate and a decrease in the amount of heat introduced to the weldment. An X70 microalloyed steel was welded using both TSAW and CWTSAW processes. Charpy-V-notch impact testing and microhardness testing showed improvement in the HAZ mechanical properties for CWTSAW samples relative to TSAW samples. Microstructural analysis, using both optical microscopy and scanning electron microscopy (SEM), indicated the formation of finer prior austenite grains (PAG) and less M-A constituent within the CGHAZ of the CWTSAW samples. These improvements are due to lower actual heat introduced to the weldment and a relatively faster cooling rate.
A Finite Element Procedure to Model the Effect of Hydrostatic Testing on Subsequent Fatigue Crack Growth
Authors: Anderson Ted L. , Thorwald Greg V.
Published: November 2016

Hydrostatic testing of pipelines that are subject to pressure cycling and fatigue damage can alter the intrinsic characteristics of flaws that survive the test. The effect is generally favorable, as test pressures well above the maximum operating pressure (MOP) can significantly reduce the subsequent rate of fatigue crack growth. The phenomenon is known as fatigue retardation, which is caused by crack closure due to compressive residual stresses created by plastic deformation during the hydrotest.

Fatigue retardation following an overload event is a well-known phenomenon in metallic structures, but there has been little or no effort to take advantage of this beneficial effect in pipelines. This paper presents a modeling procedure aimed at quantifying fatigue retardation following a hydrostatic test. A series of 3D elastic-plastic finite element simulations have been performed to model fatigue crack growth following a pressure test. The effect of test pressure and MOP on plasticity-induced crack closure was studied. The relative effect of fatigue retardation on remaining life was demonstrated with several examples. In some cases, the results were counter intuitive.
The Short and Long Term Effects of Elevated Temperature on the Mechanical Properties of Line Pipe Steels
Authors: Jacobs Taylor R. , Matlock David K. , Findley Kip O. , Collins Laurie
Published: November 2016

The mechanical properties of six industrially produced pipeline steels equivalent to API X52, X60, and X70 specifications were evaluated in the temperature range of 200–350 °C. The steels were tested in uniaxial tension at strain rates of 10−4 and 8 × 10−4 s−1 in the as-received condition and after a low temperature 100 h aging process under a 419 MPa tensile stress. Dynamic strain aging was identified in the tensile data with the observation of serrated yielding, minima in ductility and maxima in ultimate tensile strength with respect to temperature. In addition to minima in ductility, higher strength grade steels exhibited maxima in ductility at high temperatures and greater amounts of strengthening compared to the lower strength grade, both which could be attributed to the precipitation of carbides or nitrides during tensile deformation. The low temperature aging process resulted in increased yield strength due to static strain aging, slight changes to ultimate tensile stress and, no observable change in ductility. Thus, based on the results discussed it is suggested that pipeline steels can be designed based on room temperature tensile properties, using established corrections for such properties at elevated temperatures.
Fracture Toughness of Welds Using As-Welded-Geometry/SENT Specimens
Authors: Uddin Mohammed , Wilkowski Gery
Published: November 2016

The single-edge notch tension (SENT) test is frequently used for the assessment of the integrity of welds with flaws in them; this is done since the SENT specimen has the same flaw orientation as a surface flaw in the weld, and has similar constraint that affects the brittle-to-ductile transition and upper-shelf value. Traditionally SENT specimens are machined with a rectangular cross-section from the weld, and the thickness might be reduced because of that machining operation. The toughness value of the constant thickness machined specimen is then used in a pipe fracture analysis. Of course real welds have crowns in the roots of the weldment, which are ignored in both the fracture specimen test and the pipe geometry fracture analysis. To assess the importance of the weld crown and root, SENT tests were conducted as an exploratory aspect to determine the effect on toughness. Additionally, assessment of results where the SENT specimen breaks in the weld or base metal outside the weld due to the reinforcing that is usually machined off and ignored were conducted. The use of a full-weldment cross-section in SENT testing can be done for axial seam welds or circumferential welds. The initial work was on axial seam welds, although there is ongoing work for circumferential welds as well.
Update of Understanding of Near-Neutral pH SCC Crack Growth Mechanisms and Development of Pipe-Online Software for Pipeline Integrity Management
Authors: Chen Weixing , Zhao Jiaxi , Been Jenny , Chevil Karina , Van Boven Greg , Keane Sean
Published: November 2016

This paper is aimed to introduce Pipe-Online Software that has been developed recently for crack growth and remaining service life prediction for pipelines experiencing near-neutral pH stress corrosion cracking and corrosion fatigue. The software was developed based on the latest understanding of the physical, chemical and mechanical processes involved during crack initiation, early crack growth and coalescence, and stage II crack growth. In each stage of cracking, governing equations were established based on extensive experimental simulations under realistic conditions found during pipeline operation in the field and vast amounts of field data collected, which include pipeline steel properties, crack geometries, field environmental conditions, Supervisory Control and Data Acquisition (SCADA) data of oil and gas pipelines. The model has considered a wide range of conditions that could lead to the crack initiation, crack dormancy and crack transition from a dormant state to active growth. It is concluded that the premature rupture caused by stress cracking at a service life of about 20–30 years commonly found during field operation could take place only when all the worst conditions responsible for crack initiation and growth have been realized concurrently at the site of rupture. This also explains the reason why over 95% of near-neutral pH cracks remain harmless, while about 1% of them become a threat to the integrity of pipeline steels. It has been found that crack initiation and early stage crack growth are primarily caused by the direct dissolution of steels at constrained areas. The rate of dissolution can be high at the pipe surface because of various galvanic effects, but decreases to a low value as the cracks approach a depth of ∼ 1.0 mm, leading to a state of dormancy as generally observed in the field. In stage II crack growth, the software has considered loading interactions occurring during oil and gas pipeline operations with underload-type variable pressure fluctuations. The software has provided predicted lifetimes that are comparable to the actual service lives found in the field. This forms a sharp contrast with the predictions made by existing methods that are generally conservative or inconsistent with the field observations.
Application of Thermal Analysis Tool for Girth Welding Procedure Qualification
Authors: Dekhane Aditya , Wang Alex , Wang Yong-Yi , Quintana Marie
Published: November 2016

The mechanical properties of welds are governed by the final microstructure that develops as an interaction between the chemical composition and cooling rates produced by welding thermal cycles. For welds in modern microalloyed thermomechanically controlled processed (TMCP) pipeline steels, the microstructure and mechanical properties can be extremely sensitive to cooling rates. The development and qualification of welding procedures to achieve targeted mechanical properties is often an iterative process. Accurate knowledge of welding thermal cycles and cooling rates as a function of welding parameters is valuable for optimization of welding process development.

This paper covers the development, validation, and application of a girth welding thermal analysis tool. The core of the tool is a numerical model that has a two-dimensional, axi-symmetrical finite element procedure to simulate the transient heat transfer processes both in the weld metal and the heat affected zone (HAZ). The tool takes welding parameters, pipe and bevel geometry, and thermal properties as inputs and predicts thermal cycles and cooling rates in weld metal and HAZ. The comparison of thermal cycles between experimental measurements and the model predictions show the tool was robust and accurate.

This tool is particularly effective in understanding the thermal history and resulting microstructure and mechanical properties of welds produced with high-productivity gas metal arc welding (GMAW), such as mechanized dual-torch pulsed gas metal arc welding (DT GMAW-P). The tool was used in optimization of development and qualification of welding procedures of a DT GMAW-P process under a tight time schedule. The actual welds were fabricated according to the optimized welding procedures followed by the mechanical testing of welds. Good agreement was found between the predicted tensile properties and those from experimental tests. The welding procedures were qualified within the tight time schedule by avoiding iterative trials, and reducing the cost associated with the making of trial welds and mechanical testing by approximately 50%. This tool has also been applied in the application of essential welding variables methodology (EWVM) for X80 and X70 linepipe steels [1, 2].

Future applications of the tools include the revamp of the approach to essential variables in welding procedure qualification. In particular, the parameters affecting cooling rates may be “bundled” together towards the one critical factor affecting weld properties, i.e., cooling rate. The individual parameters may be varied beyond the limits in the current codes and standards as long as their combined effects make the cooling rate stay within a narrow band. It is expected that the same framework of approaches to GMAW processes can be extended other welding processes, such as FCAW and SMAW.
Bim+ ion beam patterning of germanium surfaces at different temperatures and ion fluence
Authors: Hernández Angélica Guadalupe , Kudriavtsev Yuriy
Published: November 2016

Studies of pattern formation on variable-temperature Ge targets by ion beam sputtering are presented. A high-energy heavy polyatomic bismuth ion beam was used to reveal the effect of thermal spikes in the dynamics of pattern formation. By varying the target temperature in the range from 123 to 773 K, different morphologies of the irradiated surfaces were obtained. A smooth surface was observed for a target temperature of 123 K. Sputtering at a target temperature between 300 and 573 K gave rise to poorly oriented dot patterns and resulted in a checkerboard pattern at a target temperature of 773 K. Mechanisms of surface patterning under ion irradiation are discussed.
Local attenuation curve optimization framework for high quality perfusion maps in low‐dose cerebral perfusion CT
Authors: Van Nieuwenhove Vincent , Van Eyndhoven Geert , Batenburg K. Joost , Buls Nico , Vandemeulebroucke Jef , De Beenhouwer Jan , Sijbers Jan
Published: November 2016

Purpose:

Cerebral perfusion x‐ray computed tomography (PCT) is a powerful tool for noninvasive imaging of hemodynamic information throughout the brain. Conventional PCT requires the brain to be imaged multiple times during the perfusion process, and hence radiation dose is a major concern. The authors propose a PCT reconstruction algorithm that allows for lowering the dose while maintaining a high quality of the perfusion maps. It relies on an accurate estimation of the arterial input function (AIF), which in turn depends on the quality of the attenuation curves in the arterial region.

Methods:

The authors propose the local attenuation curve optimization (LACO) framework. It accurately models the attenuation curves inside the vessel and arterial regions and optimizes its shape directly based on the acquired x‐ray projection data.

Results:

The LACO algorithm is extensively validated with simulation and real clinical experiments. Quantitative and qualitative results show that our proposed approach accurately estimates the vessel and arterial attenuation curves from only few x‐ray projections. In contrast to conventional approaches, where the AIF is estimated based on the reconstructed images, our method computes an optimal AIF directly based on the projection data, resulting in far more accurate perfusion maps.

Conclusions:

The LACO algorithm allows estimating high quality perfusion maps in low dose scanning protocols.
Computer aided prognosis for cell death categorization and prediction in vivo using quantitative ultrasound and machine learning techniques
Authors: Gangeh M. J. , Hashim A. , Giles A. , Sannachi L. , Czarnota G. J.
Published: November 2016

Purpose:

At present, a one‐size‐fits‐all approach is typically used for cancer therapy in patients. This is mainly because there is no current imaging‐based clinical standard for the early assessment and monitoring of cancer treatment response. Here, the authors have developed, for the first time, a complete computer‐aided‐prognosis (CAP) system based on multiparametric quantitative ultrasound (QUS) spectroscopy methods in association with texture descriptors and advanced machine learning techniques. This system was used to noninvasively categorize and predict cell death levels in fibrosarcoma mouse tumors treated using ultrasound‐stimulated microbubbles as novel endothelial‐cell radiosensitizers.

Methods:

Sarcoma xenograft tumor‐bearing mice were treated using ultrasound‐stimulated microbubbles, alone or in combination with x‐ray radiation therapy, as a new antivascular treatment. Therapy effects were assessed at 2–3, 24, and 72 h after treatment using a high‐frequency ultrasound. Two‐dimensional spectral parametric maps were generated using the power spectra of the raw radiofrequency echo signal. Subsequently, the distances between “pretreatment” and “post‐treatment” scans were computed as an indication of treatment efficacy, using a kernel‐based metric on textural features extracted from 2D parametric maps. A supervised learning paradigm was used to either categorize cell death levels as low, medium, or high using a classifier, or to “continuously” predict the levels of cell death using a regressor.

Results:

The developed CAP system performed at a high level for the classification of cell death levels. The area under curve of the receiver operating characteristic was 0.87 for the classification of cell death levels to both low/medium and medium/high levels. Moreover, the prediction of cell death levels using the proposed CAP system achieved a good correlation (r = 0.68,  p < 0.001) with histological cell death levels as the ground truth. A statistical test of significance between individual treatment groups with the corresponding control group demonstrated that the predicted levels indicated the same significant changes in cell death as those indicated by the ground‐truth levels.

Conclusions:

The technology developed in this study addresses a gap in the current standard of care by introducing a quality control step that generates potentially actionable metrics needed to enhance treatment decision‐making. The study establishes a noninvasive framework for quantifying levels of cancer treatment response developed preclinically in tumors using QUS imaging in conjunction with machine learning techniques. The framework can potentially facilitate the detection of refractory responses in patients to a certain cancer treatment early on in the course of therapy to enable switching to more efficacious treatments.
Modeling and design of two-dimensional membrane-type active acoustic metamaterials with tunable anisotropic density
Authors: Allam Ahmed , Elsabbagh Adel , Akl Wael
Published: November 2016

A two-dimensional active acoustic metamaterial with controllable anisotropic density is introduced. The material consists of composite lead–lead zirconate titanate plates clamped to an aluminum structure with air as the background fluid. The effective anisotropic density of the material is controlled, independently for two orthogonal directions, by means of an external static electric voltage signal. The material is used in the construction of a reconfigurable waveguide capable of controlling the direction of the acoustic waves propagating through it. An analytic model based on the acoustic two-port theory, the theory of piezoelectricity, the laminated pre-stressed plate theory, and the S-parameters retrieval method is developed to predict the behavior of the material. The results are verified using the finite element method. Excellent agreement is found between both models for the studied frequency and voltage ranges. The results show that, below 1600 Hz, the density is controllable within orders of magnitude relative to the uncontrolled case. The results also suggest that simple controllers could be used to program the material density toward full control of the directivity and dispersion characteristics of acoustic waves.
Incorporating explicit geospatial data shows more species at risk of extinction than the current Red List
Authors: Ocampo-Peñuela Natalia , Jenkins Clinton N. , Vijay Varsha , Li Binbin V. , Pimm Stuart L.
Published: November 2016

The Red List classification of species’ risks of extinction has seriously underestimated the risks of many species.
Trained Immunity and Susceptibility to HIV
Authors: Derrick Steven C.
Published: November 2016

In this issue of
Clinical and Vaccine Immunology
, K. Jensen et al. (Clin Vaccine Immunol 24:e00360-16, 2017,
https://doi.org/10.1128/CVI.00360-16
) describe a dual-purpose attenuated
Mycobacterium tuberculosis-
simian immunodeficiency virus vaccine (AMTB-SIV). Interestingly, immunized infant macaques required fewer oral exposures to SIV to become infected relative to nonimmunized animals. The authors hypothesized that augmented susceptibility to SIV was due to activation of CD4
+
T cells through trained immunity. This commentary explores the possible relationship between trained immunity, enhanced CD4 T cell responses, and increased susceptibility to human immunodeficiency virus (HIV).
The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa
Authors: Huang H , Hancock R E
Published: November 2016

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane. In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein. Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS. When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel. From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 microM. In contrast, no decrease in channel conductance was observed for the OprDdeltaL2 channel upon addition of up to 2.4 microM imipenem, confirming that external loop 2 was involved in imipenem binding. Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to beta-lactams, quinolones, chloramphenicol, and tetracycline. Planar lipid bilayer analysis indicated that the OprDdeltaL5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site. The disposition of these loop regions in the interior of the OprD channel is discussed.
Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins
Authors: Barloy F , Delécluse A , Nicolas L , Lecadet M M
Published: November 2016

A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.
Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites
Authors: Jiang H , Parales R E , Lynch N A , Gibson D T
Published: November 2016

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.
The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production
Authors: Lomovskaya N , Hong S K , Kim S U , Fonstein L , Furuya K , Hutchinson R C
Published: November 2016

The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.
Elements of signal transduction in Mycobacterium tuberculosis: in vitro phosphorylation and in vivo expression of the response regulator MtrA
Authors: Via L E , Curcic R , Mudd M H , Dhandayuthapani S , Ulmer R J , Deretic V
Published: November 2016

A putative two-component system, mtrA-mtrB, was isolated from M. tuberculosis H37Rv by using phoB from Pseudomonas aeruginosa as a hybridization probe. The predicted gene product of mtrA displayed high similarity with typical response regulators, including AfsQ1, PhoB, PhoP, and OmpR. The predicted gene product of mtrB displayed similarities with the histidine protein kinases AfsQ2, PhoR, and EnvZ and other members of this class of proteins. Expression analysis in the T7 system showed that mtrA encoded a polypeptide with an apparent molecular mass of 30 kDa. MtrA was overproduced, purified, and demonstrated to participate in typical phosphotransfer reactions using a heterologous histidine protein kinase, CheA, as a phosphoryl group donor. Mycobacterium bovis BCG, harboring an mtrA-gfp (green fluorescent protein cDNA) transcriptional fusion, was used to monitor mtrA expression in infected J774 monolayers. Flow cytometric and fluorescence microscopic analyses indicated that the mtrA promoter was activated upon entry and incubation in J774 macrophages. In contrast, the hsp60-gfp fusion displayed no change in expression under the growth conditions tested. These results suggest a potential role for mtrA in adaptation of the M. tuberculosis complex organisms to environmental changes which may include intracellular conditions.
Site-directed mutational analysis of the osmotically regulated proU promoter of Salmonella typhimurium
Authors: Zhang X , Fletcher S A , Csonka L N
Published: November 2016

We carried out PCR mutagenesis of the proU promoter of Salmonella typhimurium, in order to identify sequences important for its osmotic control. We obtained five mutations in the -35 element: two decreased the promoter strength, one increased it, and the others had no effect. However, none abolished osmotic control, suggesting that the sequence of the -35 element is not crucial for osmotic control.
The tamA gene of Aspergillus nidulans contains a putative zinc cluster motif which is not required for gene function
Authors: Davis M A , Small A J , Kourambas S , Hynes M J
Published: November 2016

Expression of many nitrogen catabolic enzymes is controlled by nitrogen metabolite repression in Aspergillus nidulans. Although the phenotypes of tamA mutants have implicated this gene in nitrogen regulation, its function is unknown. We have cloned the tamA gene by complementation of a new tamA allele. The tamA sequence shares significant homology with the UGA35/DAL81/DURL gene of Saccharomyces cerevisiae. In vitro mutagenesis of sequences encoding a putative zinc cluster DNA binding domain indicated that this motif is not required for in vivo TamA function.
Effects of mecA and mecB (clpC) mutations on expression of sigD, which encodes an alternative sigma factor, and autolysin operons and on flagellin synthesis in Bacillus subtilis
Authors: Rashid M H , Tamakoshi A , Sekiguchi J
Published: November 2016

The expression of the major vegetative phase-specific autolysin genes (cwlB [lytC] and cwlG [lytD]) was greatly reduced by mecA and mecB null mutations. In contrast to the negative effects on late competence genes (such as comG) and levansucrase gene (sacB) expression, this positive effect of mec genes on autolysin gene expression was not mediated through the ComK protein but apparently through the level of the SigD protein. The pleiotropic effects of the mec mutations, i.e., the reduction of sigD expression and the overexpression of the ComK protein, seem not to be interwoven since the SigD- and ComK-dependent functions are clearly separable in the mec mutants. We also show that the synthesis of the flagellin protein, which is encoded by the SigD-dependent hag gene, was similarly affected by the mec mutations. Complementation analysis with a SigD-overproducing plasmid, pHYSigD, in mec mutants revealed the reversion of almost all of the SigD-dependent phenotypes except motility. This finding suggested that Mec proteins act on motility genes at two levels, one of which is apparently SigD independent. Finally, we discuss the transcriptional regulation of the sigD gene by multiple regulators, i.e., MecA, MecB, SinR (FlaD), and DegS-DegU, and its implications for cells in a global context.
Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi
Authors: Skare J T , Champion C I , Mirzabekov T A , Shang E S , Blanco D R , Erdjument-Bromage H , Tempst P , Kagan B L , Miller J N , Lovett M A
Published: November 2016

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.
2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase
Authors: Suen W C , Haigler B E , Spain J C
Published: November 2016

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.
Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): genes, gene products, and transcripts of the arcRACB gene cluster
Authors: Ruepp A , Soppa J
Published: November 2016

Fermentative growth via the arginine deiminase pathway is mediated by the enzymes arginine deiminase, carbamate kinase, and catabolic ornithine transcarbamylase and by a membrane-bound arginine-ornithine antiporter. Recently we reported the characterization of catabolic ornithine transcarbamylase and the corresponding gene, arcB, from Halobacterium salinarium (formerly Halobacterium halobium). Upstream of the arcB gene, three additional open reading frames with halobacterial codon usage were found. They were identified as the arcC gene coding for carbamate kinase, the arcA gene coding for arginine deiminase, and a gene, tentatively termed arcR, coding for a putative regulatory protein. The identification of the arcC and arcA genes was verified, respectively, by heterologous expression of the enzyme in Haloferax volcanii and by protein isolation and N-terminal sequence determination of three peptides. The gene order arcRACB differs from the gene order arcDABC in Pseudomonas aeruginosa, the only other organism for which sequence information is available. Transcripts from H. salinarium cultures grown fermentatively or aerobically were characterized by Northern (RNA) blot and primer extension analyses. It was determined (i) that monocistronic transcripts corresponding to the four open reading frames exist and that there are three polycistronic transcripts, (ii) that the level of induction during fermentative growth differs for the various transcripts, and (iii) that upstream of the putative transcriptional start sites for the three structural genes there are sequences with similarities to the halobacterial consensus promoter. The data indicate that expression of the arc gene cluster and its regulation differ in H. salinarium and P. aeruginosa.
Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydroorotate dehydrogenase activity in Bacillus subtilis
Authors: Kahler A E , Switzer R L
Published: November 2016

An in-frame deletion in the coding region of a gene of previously unidentified function (which is called orf2 and which we propose to rename pyrDII) in the Bacillus subtilis pyr operon led to pyrimidine bradytrophy, markedly reduced dihydroorotate dehydrogenase activity, and derepressed levels of other enzymes of pyrimidine biosynthesis. The deletion mutation was not corrected by a plasmid encoding pyrDI, the previously identified gene encoding dihydroorotate dehydrogenase, but was complemented by a plasmid encoding pyrDII. We propose that pyrDII encodes a protein subunit of dihydroorotate dehydrogenase that catalyzes electron transfer from the pyrDI-encoded subunit to components of the electron transport chain.
A methyl-accepting protein involved in multiple-sugar chemotaxis by Cellulomonas gelida
Authors: Hsing W , Canale-Parola E
Published: November 2016

Tethered-cell and capillary assays indicated that L-methionine is required by Cellulomonas gelida for its normal cell motility pattern and chemotaxis and that S-adenosylmethionine is involved in sugar chemotaxis by this cellulolytic bacterium. In addition, in vivo methylation assays showed that several proteins were methylated in the absence of protein synthesis. The incorporated methyl groups were alkali sensitive. Of special interest was the observation that the methylation level of a 51,000-Mr protein increased two- to fivefold upon addition of various sugar attractants and decreased after the removal of the attractants. The increase was less pronounced in mutants defective in sugar chemotaxis and appeared to be specifically involved with sugar chemotaxis. Furthermore, cell fractionation and in vitro methylation assays demonstrated that the 51,000-Mr protein is located in the cytoplasmic membrane. These results suggest that a specific methyl-accepting chemotaxis protein is involved in multiple-sugar chemotaxis by C gelida. During chemotaxis, the changes of methylesterase activity in C gelida cells were similar to those in Escherichia coli RP437 cells, as determined by a continuous-flow assay for methanol evolution. Thus, the mechanism of methyl-accepting chemotaxis protein-mediated chemotaxis of the gram-positive C. gelida appears to be similar to that of the gram-negative E. coli rather than to that of other gram-positive bacteria, such as Bacillus subtilis.
Isolation, purification, and in vitro characterization of recessive-lethal-mutant RNA polymerases from Escherichia coli
Authors: Tavormina P L , Landick R , Gross C A
Published: November 2016

The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.
Experimental rejection of a nonadaptive explanation for increased cell size in Escherichia coli
Authors: Mongold J A , Lenski R E
Published: November 2016

Populations of Escherichia coli that have been serially propagated for thousands of generations in glucose minimal medium show heritable increases in both cell size and growth rate. We sought to test the hypothesis that the increased cell size of the derived genotypes could be explained solely by their faster growth. The regression of cell size on growth rate differed significantly between populations having ancestral and derived genotypes, with the latter producing larger cells over almost the entire range of growth rates. Thus, the physiological coupling between cell size and growth rate has been evolutionarily altered.
The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins
Authors: Bailey D A , Feldmann P J , Bovey M , Gow N A , Brown A J
Published: November 2016

A hyphally regulated gene (HYR1) from the dimorphic human pathogenic fungus Candida albicans was isolated and characterized. Northern (RNA) analyses showed that the HYR1 mRNA was induced specifically in response to hyphal development when morphogenesis was stimulated by serum addition and temperature elevation, increases in both culture pH and temperature, or N-acetylglucosamine addition. The HYR1 gene sequence revealed a 937-codon open reading frame capable of encoding a protein with an N-terminal signal sequence, a C-terminal glycosylphosphatidylinositol-anchoring domain, 17 potential N glycosylation sites, and a large domain rich in serine and threonine (51% of 230 residues). These features are observed in many yeast cell wall proteins, but no homologs are present in the databases. In addition, Hyr1p contained a second domain rich in glycine, serine, and asparagine (79% of 239 residues). The HYR1 locus in C. albicans CAI4 was disrupted by "Ura-blasting," but the resulting homozygous delta hyr1/delta hyr1 null mutant displayed no obvious morphological phenotype. The growth rates for yeast cells and hyphae and the kinetics of germ tube formation in the null mutant were unaffected. Aberrant expression of HYR1 in yeast cells, when an ADH1-HYR1 fusion was used, did not stimulate hyphal formation in C. albicans or pseudohyphal growth in Saccharomyces cerevisiae. HYR1 appears to encode a nonessential component of the hyphal cell wall.