bluuebunny/crossref_metadata_2025 huggingface dataset:
A Study of Crack Detection Ultrasonic Attributes to Manage Leak Threats Associated With Short Crack-Like Flaws in ERW Pipelines
Authors: Korol Kaitlyn , Hubert Yvan , Fredine Gordon , Senf Petra , Koon Koon Sherry-Ann
Published: November 2016

Inline Inspection (ILI) tools along with hydrostatic testing have been the primary identification and mitigation techniques for cracking threats on liquids pipelines. Each technique faces detection challenges in relation with the weld type, geometry, and feature types, sizes and orientations. Low frequency electric resistance welds (LF ERWs) are subject to a number of crack-like defects due to the ERW manufacturing process. These defects may include fatigue cracks, lack of fusion, burned metal defects, stitched welds, cold welds, cracks in hard HAZ, surface breaking hook cracks near the weld and selective seam corrosion [1].

Within a population of features in a pipeline, a subpopulation can exist of short, deep defects (>50% wt) that may be undersized by the ILI tool or not detected by a hydrostatic test due to the length of the flaw. For ILI tools, a length detection threshold is set based on the tool speed (which is dictated by the tool type and configuration). A feature may be undersized by the ILI tool if its length is below this tool threshold. For hydrostatic testing, through-wall flaws may be undetected if the flaw length is below the critical length for a significant leak. Through detailed ILI data analysis, Enbridge along with PII Pipeline Solutions has been able to consistently identify short and deep crack-related defects on LF ERW pipe through means other than feature dimensions provided by the ILI tool.

In-ditch non-destructive examination and destructive laboratory testing has confirmed these features are critical and fall below current ILI tool’s detection thresholds. This paper discusses unique ILI data attributes that may identify a more severe feature than would conventional ILI sizing practices, and how the identification and selection procedure is being applied across Enbridge’s pipeline system. This analysis effort aligns with Enbridge’s goal to continuously improve its integrity management processes and further enhance the safety of its pipelines.
Modified Two-Curve Model for Predicting Fracture Arrest Toughness and Arrest Distance of Full-Size Burst Tests
Authors: Zhu Xian-Kui
Published: November 2016

Running fracture control is a very important technology for gas transmission pipelines with large diameter and high pressure. The Battelle two-curve (BTC) model developed in the early 1970s has been widely used in pipeline industry to determine arrest toughness in terms of the Charpy energy. Because of its semi-empirical nature and calibration with test data only for grades up to X65, the BTC does not work for higher grades. Simple corrections were thus proposed to extend the BTC model to higher grades, but limited to those grades considered. Moreover, the BTC model only predicts the minimum arrest toughness, but not arrest distance.

To fill the technical gaps, this paper proposes a modified two-curve (MTC) model and a fracture arrest distance model in reference to the Charpy energy. The MTC model coupling with an arrest distance algorithm can predict fracture arrest toughness and arrest distance in one simulation of numerical integration for a single pipe or a set of multiple pipes with given toughness. Two sets of full-scale burst test data for X70 and X80 are used to validate the proposed model, and the results show good agreements between the predictions and full-scale test data of arrest toughness and arrest distance as well. The MTC model is then applied to optimize a design of pipe segment arrangements for a mockup full-scale burst test on a high-strength pipeline steel. The MTC simulation results confirm the experimental observation that different pipe arrangements determine different arrest toughness and arrest distance for the same grade pipes.
Review of Engineering Fracture Mechanics Model for Pipeline Applications
Authors: Polasik Steven J. , Jaske Carl E. , Bubenik Thomas A.
Published: November 2016

Operating pipelines may contain crack-like flaws created during fabrication or induced by service. Stress-corrosion cracking (SCC) and fatigue are two common mechanisms that cause cracks to develop in operating pipelines. Engineering fracture mechanics models are typically used to assess the potential for crack-like flaws to result in pipeline failure. To this end, an inelastic fracture mechanics model was developed and incorporated into the CorLAS™ computer program that is used by many pipeline operators. This paper reviews and documents the details of the fracture mechanics model. It provides the equations used to compute the parameters in the model and discusses their engineering basis. Correlations of predictions made using the model with the results of tests and pipeline failures are presented. Typical applications of the model are also reviewed. Finally, areas of possible improvements are discussed.
Advanced Integrity Management Framework for Pipelines
Authors: LeBlanc Len , Kresic Walter , Keane Sean , Munro John
Published: November 2016

This paper describes the integrity management framework utilized within the Enbridge Liquids Pipelines Integrity Management Program. The role of the framework is to provide the high-level structure used by the company to prepare and demonstrate integrity safety decisions relative to mainline pipelines, and facility piping segments where applicable. The scope is directed to corrosion, cracking, and deformation threats and all variants within those broad categories. The basis for the framework centers on the use of a safety case to provide evidence that the risks affecting the system have been effectively mitigated. A ‘safety case’, for the purposes of this methodology is defined as a structured argument demonstrating that the evidence is sufficient to show that the system is safe.[1]

The decision model brings together the aspects of data integration and determination of maintenance timing; execution of prevention, monitoring, and mitigation; confirmation that the execution has met reliability targets; application of additional steps if targets are not met; and then the collation of the results into an engineering assessment of the program effectiveness (safety case). Once the program is complete, continuous improvement is built into the next program through the incorporation of research and development solutions, lessons learned, and improvements to processes.

On the basis of a wide range of experiences, investigations and research, it was concluded that there are combinations of monitoring and mitigation methods required in an integrity program to effectively manage integrity threats. A safety case approach ultimately provides the structure for measuring the effectiveness of integrity monitoring and mitigation efforts, and the methodology to assess whether a pipeline is sufficiently safe with targets for continuous improvement. Hence, the need for the safety case is to provide transparent, quantitative integrity program performance results which are continually improved upon through ongoing revalidations and improvement to the methods utilized. This enables risk reduction, better stakeholder awareness, focused innovation, opportunities for industry information sharing along with other benefits.
Comparison of J-Integral Measurement Methods on Clamped Single-Edge Notched Tension Specimens
Authors: Weeks Timothy S. , Sowards Jeffrey W. , Rentz Ross A. , Read David T. , Lucon Enrico
Published: November 2016

This paper reports an extension of a previous study that compared methods of evaluating J by the crack mouth opening displacement and by surface strain gradients. Here, the surface strain gradients are measured by three-dimensional digital image correlation. The results herein represent a small test matrix that involved evaluation of the J-integral for clamped single-edge notched tensile specimens from API 5L X65 base-metal, weld metal and the adjacent heat affected zone; the J-integral was evaluated by a standardized procedure utilizing the crack mouth opening displacement (CMOD) and by the contour integral method on an external surface strain contour. Digital image correlation provides sufficient full-field strain data for use by this method and is considerably more robust than surface-mounted strain gage instrumentation. A series of validity checks are presented that demonstrate that the data are useful and valuable. Experimental determination of the J-integral is not limited to thoroughly analyzed test geometries and may be achieved with limited instrumentation. Furthermore, the method described does not require a determination of crack size nor any instrumentation that requires access to the crack mouth.
The Benefits of Accurate ILI Performance on Pipeline Integrity Programs for Axial Crack and Metal Loss Corrosion Threats
Authors: Adianto Riski H. , Skow Jason B. , Sutherland Jeffrey
Published: November 2016

An analysis describing the benefits of an accurate in-line inspection (ILI) system performance is presented in this paper. A good ILI performance is characterized as an accurate description of the condition of an inspected pipeline. Information from a better ILI performance, as compared to a poorer one, can be used to reduce the number of required digs and/or extend the re-inspection interval without compromising the pipeline’s integrity. As a result, these parameters can be used to assess the benefits of the improved inspection performance.

For this analysis, the ILI performance was represented by the depth sizing accuracy as the depth of a feature is one of the most critical parameters in assessing the pressure containment capacity at the feature location. This work utilized a sensitivity analysis in which the impact of various levels of ILI performance on a pipeline integrity program in terms of the number of required repairs for a given reliability threshold was examined. The number of required repairs associated with each inspection performance was calculated using a reliability based assessment method. This method was selected because it fully accounts for the statistical characteristic of the ILI performance.

The sensitivity analysis considered two pipeline condition scenarios for two pipeline systems. The first pipeline condition scenario was characterized as having a high number of features, many of which were severe in size, while the second condition scenario consisted of fewer features that were less significant in size. The analysis was carried out for both axial stress corrosion cracking and metal loss corrosion features.

The results of the analysis show that a more accurate ILI depth measurement leads to a more accurate pipeline reliability estimate, and therefore, a reduction in the number of required repairs. However, the benefit associated with continued ILI measurement accuracy improvement exhibits a diminishing trend.
Finite Element Analysis and Test Verification of Composite Repair Length on Corrosion Defects
Authors: Mally Timothy , Keller Michael W. , Kiani Mahdi , Walker Roger , Burnworth Christopher W.
Published: November 2016

Engineered composite repairs are rapidly gaining traction as an alternative pipeline integrity repair method. These repair systems are being used to repair corrosion, erosion, dents, wrinkle bends, mechanical damage, and other anomalies that commonly occur on pipelines. Composites possess many significant advantages when compared to traditional metallic repairs. One of the most critical benefits of composites is that most composite repairs are fabricated on-site by installing an uncured composite that subsequently cures. This cure-in-place approach enables the production of repairs that can conform to complex geometries. Conformability allows for the reinforcement of damaged piping and pressure vessels that were difficult, or impossible, to repair with rigid metallic sleeves. While composites offer improved performance and reduced installation times for complex geometries, the engineering and installation of these repair systems is not as straightforward. Many areas of the methodology used to design composite repair systems are active areas of research. One of the current areas of discussion is the distance a repair must extend past a non-through-wall defect. The current nonmetallic repair standards, ASME PCC-2 Article 4.1 [1] and ISO/TS 24817 [2], suggest that a composite repair should extend a varying length past the defect in each direction, based on either the diameter and thickness of the pipe to be repaired or the stiffness of the composite. For the case of through-wall defects, the design methodology is based on the lap-shear strength of the composite and generally leads to repairs that extend farther beyond the defect when compared with non-through wall repiars. For this damage scenario, the presence of an extended repair area is considered an additional factor of safety. However, for non-leaking external defects, there is some debate as to whether the standard-prescribed, minimum repair length is required. This paper investigates a comparison of the minimum repair length required by ASME PCC-2 Equation 17 versus a shorter overlap length of 2 inches. Finite Element Analysis is used to model the difference in the two repair approaches. The FEA model is then verified by a hydrotest on a full-scale spool repair. Six specimens are machined to 75% wall loss and repaired with an engineered composite solution. The defect design for these repairs is adopted from guidance in ASME PCC-2. After installation of the repair, the pipe spool is then subjected to a hydrotest to 100% SMYS. After the 100% SMYS test, the specimens were ruptured in order to compare the ultimate strengths of the two repair approaches. All test results are compared using appropriate statistical approaches to determine significance (α = 0.05).
The Influence of the Bourdon Effect on Pipe Elbow
Authors: Abdulhameed Diana , Adeeb Samer , Cheng Roger , Martens Michael
Published: November 2016

Pipe elbows are frequently used in a pipeline system to change the directions. Thermal expansion and internal pressure results in bending moments on the bends causing ovalization of the initial circular cross-section. The ability of the bend to ovalize will result in an increase in the bend flexibility when compared to straight pipes [1]. In case of bends subjected to internal pressure, the pipe will start to straighten out due to the difference between the intrados and extrados surface areas. The internal pressure causes unbalanced thrust forces tending to open up the elbow depending on its stiffness and surrounding constraints. These forces tending to cause ovalization of the cross section and causing the tendency of pipe bends to open up are termed the “Bourdon effect”. If these unbalanced thrust forces are not taken into consideration, unanticipated deformations and high stress levels could occur at the elbow location that may not be accounted for in traditional stress analysis [2]. A better understanding of the influence of the Bourdon effect on the elbow design parameters is required. Past studies have investigated the behaviour of pipe elbows under closing bending moment and proposed factors that account for the increased flexibility and high stress levels resulted from ovalization. These factors are used in the current design codes [3],[4] &[5] and known as the flexibility factor and stress intensification factor.

In this investigation, pipe elbows with different nominal pipe size and various bend radiuses to internal pipe radius ratios (R/r) are studied to get a better understanding of the Bourdon effect and its influence on the pipe stresses and deformations. Differential equilibrium equations are solved to derive a mathematical model to evaluate the unbalanced thrust forces resulted from the Bourdon effect on a pipe elbow. The forces evaluated from the derived model are compared to finite element model results and showed excellent agreement. A comparison between the CSA-Z662 code and the FEA results is conducted to investigate the applicability of the stress intensification factors used in the current design code for different loading cases. The study showed that the external bending moment direction acting on the pipe has a significant effect on the distribution of stresses on the pipe elbow and significantly depending on the level of applied internal pressure.
Evaluating Pipeline Ovality Acceptability Criteria for Straight Pipe Sections
Authors: Kemp David , Gossard Justin , Finneran Shane , Bratton Joseph
Published: November 2016

Pipe ovalization, a deviation from the circular nominal cross section, is a common occurrence during the manufacturing of pipe sections. Additionally, ovalization can also occur in pipelines during and after installation and construction. CSA Z662-11 [1] provides an acceptance criteria of 5% for pipeline ovality in bends, however there is a variation in acceptance criteria for pipe ovality occurring in straight pipe sections. An industry review of pipeline design, operation, and maintenance codes was conducted to determine the industry acceptance for ovality limits in straight pipe sections. Based upon this industry review, the ovality limits were evaluated against constructability limits, limitations for passage of in-line-inspection (ILI) tools, as well as evaluating the stress in an ovalized pipe section compared to the maximum allowable stress of the pipe. During this review, it was revealed that allowable stress was the limiting factor for pipeline ovality, compared to constructability and ILI tool passage, thus this paper primarily discusses limitations related to remaining strength for ovalized pipe sections. The API 579 Fitness-for-Service assessment was used to evaluate varying levels of ovality to determine acceptability criteria for ovalization in straight pipe. The criteria was first established using a level 2 Fitness-for-Service assessment, which was then evaluated with a level 3 assessment using finite element analysis. This criterion was evaluated using multiple pipeline diameters and wall thickness in order to determine scalability.
Overpressure Protections in Liquid Pipeline Hydraulic Design
Authors: Zhou Alan X. L. , Yu David , Cabrejo Victor
Published: November 2016

Continuous economic development demands safe and efficient means of transporting large quantities of crude oil and other hydrocarbon products over vast extensions of land. Such transportation provides critical links between organizations and companies, permitting goods to flow between their facilities. Operation safety is paramount in transporting petroleum products in the pipeline industry. Safety can affect the performance and economics of pipeline system. Pipeline design codes also evolve as new technologies become available and management principles and practices improve. While effective operation safety requires well-trained operators, adequate operational procedures and compliance with regulatory requirements, the best way to ensure process safety is to implement safety systems during the design stage of pipeline system.

Pressure controls and overpressure protection measures are important components of a modern pipeline system. This system is intended to provide reliable control and prevent catastrophic failure of the transport system due to overpressure conditions that can occur under abnormal operating conditions. This paper discusses common pressure surge events, options of overpressure protection strategies in pipeline design and ideas on transient hydraulic analyses for pipeline systems. Different overpressure protection techniques considered herein are based on pressure relief, pressure control systems, equipment operation characteristics, and integrated system wide approach outlining complete pressure control and overpressure protection architecture for pipeline systems. Although the analyses presented in this paper are applicable across a broad range of operating conditions and different pipeline system designs, it is not possible to cover all situations and different pipeline systems have their own unique solutions. As such, sound engineering judgment and engineering principles should always be applied in any engineering design.
A Review of Rules and Analysis Models for Interaction of LF-ERW Manufacturing Linear Features in Pipelines
Authors: Li Hao (Andy) , Prescott Jonathan , Zhang Hong (Kathy) , Viveros Arturo Terron , Ferguson James
Published: November 2016

Multiple or stacked crack-like flaws can be found at longitudinal welds on vintage low frequency (LF) ERW pipeline by inspection technologies. These features may be a result of fabrication or degradation during service. The assessment of coplanar anomalies for fitness for service requires the evaluation of interaction, coalescence, and sizing of overall depth and length for multiple and/or stacked linear features. Available industrial codes provide rules to perform interaction and multiple flaw characterization; however, in some instances these general rules may result in more features requiring repairs than is actually required for a pipeline integrity program. This paper reviews interaction rules and models for linear features developed from published codes and industrial research with the objective of identifying areas for further development.
Mechanical Properties Anisotropy in X80 Line Pipes
Authors: Gervasyev Alexey , Petrov Roumen , Pyshmintsev Igor , Struin Alexey , Leis Brian
Published: November 2016

The anisotropy of tensile properties and impact toughness of X80 ferrite-bainite pipeline steels was investigated. The lowest strength values were found in longitudinal (L) direction, medium — in the direction of wall thickness (Z-direction), the highest — in transverse (T) direction. The anisotropy of the tensile properties is low and does not change significantly with the test temperature. The maximum variation in yield stress and tensile stress do not exceed the ranges of 100 MPa and 50 MPa respectively.

The anisotropy in toughness is more clearly pronounced. Impact toughness in both T and L directions is more than 250 J/cm2 at minus 20°C while in Z-direction it is less than 30 J/cm2 at the same temperature. Low Z-direction toughness determines the susceptibility of steel to splitting during ductile fracture propagation (formation of brittle cracks parallel to the rolling plane of the plate). Microstructure and crystallographic texture analyses showed that the susceptibility to splitting is controlled by a number of factors like the predominant orientation of cleavage crystallographic planes parallel to the rolling plane, morphology of the microstructure elements, and the distribution of “second” phases.
Determining the Number of Excavations Required to Confirm the Presence or Absence of SCC on a Pipeline Following an SCCDA Process
Authors: Desjardins Guy , MacKenzie John , Aguiar David
Published: November 2016

The pipeline industry has been managing the threat of Stress Corrosion Cracking (SCC) for several years using the methods developed by NACE SCCDA [1] (Stress Corrosion Cracking Direct Assessment) and ASME B31.8S [2] standards. SCCDA is a widely accepted tool for assessing the threat of Stress Corrosion Cracking in pipelines. The process utilizes data from direct examinations at excavations to validate the process as well as to address existing SCC anomalies, if found. However, neither the recommended practices nor the literature provide a usable and practical method for determining the number of excavations necessary for the excavation program based on observed results. To address this question, the Pipeline Research Council International (PRCI) sponsored a project to develop a statistically defendable procedure to determine the number of excavations which would be required to validate the SCCDA process and confirm either the presence or absence of SCC.
Technical Note: Display window setting: An important factor for detecting subtle but clinically relevant artifacts in daily CT quality control
Authors: Long Zaiyang , Bruesewitz Michael R. , Sheedy Emily N. , Powell Michele A. , Kramer Jacqualynn C. , Supalla Randall R. , Colvin Chance M. , Bechel Jessica R. , Favazza Christopher P. , Kofler James M. , Leng Shuai , McCollough Cynthia H. , Yu Lifeng
Published: November 2016

Purpose:

This study aimed to investigate the influence of display window setting on technologist performance detecting subtle but clinically relevant artifacts in daily computed tomography (CT) quality control (dQC) images.

Methods:

Fifty three sets of dQC images were retrospectively selected, including 30 sets without artifacts, and 23 with subtle but clinically relevant artifacts. They were randomized and shown to six CT technologists (two new and four experienced). Each technologist reviewed all images in each of two sessions, one with a display window width (WW) of 100 HU, which is currently recommended by the American College of Radiology, and the other with a narrow WW of 40 HU, both at a window level of 0 HU. For each case, technologists rated the presence of image artifacts based on a five point scale. The area under the receiver operating characteristic curve (AUC) was used to evaluate the artifact detection performance.

Results:

At a WW of 100 HU, the AUC (95% confidence interval) was 0.658 (0.576, 0.740), 0.532 (0.429, 0.635), and 0.616 (0.543, 0.619) for the experienced, new, and all technologists, respectively. At a WW of 40 HU, the AUC was 0.768 (0.687, 0.850), 0.546 (0.433, 0.658), and 0.694 (0.619, 0.769), respectively. The performance significantly improved at WW of 40 HU for experienced technologists (p = 0.009) and for all technologists (p = 0.040).

Conclusions:

Use of a narrow display WW significantly improved technologists’ performance in dQC for detecting subtle but clinically relevant artifacts as compared to that using a 100 HU display WW.
Radiative feedback in Helmholtz resonators with more than one opening
Authors: Dosch Hans Günter
Published: November 2016

The resonance frequency of a Helmholtz resonator with several openings is derived, taking into account the radiation feedback between the different holes. This is done under the assumption that the distance between the openings is large as compared to their linear dimensions. The effect of a finite wall thickness of the resonator is also discussed. The radiation feedback can lead to a shift of the resonance position by more than 10%.
Identification of the Rayleigh surface waves for estimation of viscoelasticity using the surface wave elastography technique
Authors: Zhang Xiaoming
Published: November 2016

The purpose of this Letter to the Editor is to demonstrate an effective method for estimating viscoelasticity based on measurements of the Rayleigh surface wave speed. It is important to identify the surface wave mode for measuring surface wave speed. A concept of start frequency of surface waves is proposed. The surface wave speeds above the start frequency should be used to estimate the viscoelasticity of tissue. The motivation was to develop a noninvasive surface wave elastography (SWE) technique for assessing skin disease by measuring skin viscoelastic properties. Using an optical based SWE system, the author generated a local harmonic vibration on the surface of phantom using an electromechanical shaker and measured the resulting surface waves on the phantom using an optical vibrometer system. The surface wave speed was measured using a phase gradient method. It was shown that different standing wave modes were generated below the start frequency because of wave reflection. However, the pure symmetric surface waves were generated from the excitation above the start frequency. Using the wave speed dispersion above the start frequency, the viscoelasticity of the phantom can be correctly estimated.
Nonlinear climate sensitivity and its implications for future greenhouse warming
Authors: Friedrich Tobias , Timmermann Axel , Tigchelaar Michelle , Elison Timm Oliver , Ganopolski Andrey
Published: November 2016

Climate sensitivity reconstructed from eight glacial cycles exhibits state dependence and confirms global warming projections.
Marine plastic debris emits a keystone infochemical for olfactory foraging seabirds
Authors: Savoca Matthew S. , Wohlfeil Martha E. , Ebeler Susan E. , Nevitt Gabrielle A.
Published: November 2016

An infochemical found on marine plastic debris elucidates a novel mechanism for plastic ingestion by marine wildlife.
Aggregate penetrance of genomic variants for actionable disorders in European and African Americans
Authors: Natarajan Pradeep , Gold Nina B. , Bick Alexander G. , McLaughlin Heather , Kraft Peter , Rehm Heidi L. , Peloso Gina M. , Wilson James G. , Correa Adolfo , Seidman Jonathan G. , Seidman Christine E. , Kathiresan Sekar , Green Robert C.
Published: November 2016

In two separate analyses, people with disease-associated mutations exhibited features of the corresponding disease more often than expected, a result that supports the use of population-based genetic screening.
How to know what we don’t
Authors: Greene Casey S.
Published: November 2016

New computational methods predict the unobserved.
On antidepressants and still feeling low
Authors: Peñagarikano Olga
Published: November 2016

Early-life stress and amygdala reactivity predict the therapeutic effect of antidepressants.
Contribution of Maternal Immunity to Decreased Rotavirus Vaccine Performance in Low- and Middle-Income Countries
Authors: Mwila Katayi , Chilengi Roma , Simuyandi Michelo , Permar Sallie R. , Becker-Dreps Sylvia
Published: November 2016

The role of maternal immunity, received by infants either transplacentally or orally from breast milk, in rotavirus vaccine (RV) performance is evaluated here. Breastfeeding withholding has no effect on vaccine responses, but higher levels of transplacental rotavirus-specific IgG antibody contribute to reduced vaccine seroconversion. The gaps in knowledge on the factors associated with low RV efficacy in low- and middle-income countries (LMIC) remain, and further research is needed to shed more light on these issues.
Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy
Authors: Müller D J , Baumeister W , Engel A
Published: November 2016

Both surfaces of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans were imaged in buffer solution by atomic force microscopy. When adsorbed to freshly cleaved mica, the hydrophilic outer surface of the HPI layer was attached to the substrate and the hydrophobic inner surface was exposed to the stylus. The height of a single HPI layer was 7.0 nm, while overlapping edges of adjacent single layers adsorbed to mica had a height of 14.7 nm. However, double-layered stacks with inner surfaces facing each other exhibited a height of 17.4 nm. These stacks exposed the outer surface to the stylus. The different heights of overlapping layers and stacks are attributed to differences in the interaction between inner and outer surfaces. At high resolution, the inner surface revealed a protruding core with a central pore connected by six emanating arms. The pores exhibited two conformations, one with and the other without a central plug. Individual pores were observed to switch from one state to the other.
Redundant homosexual F transfer facilitates selection-induced reversion of plasmid mutations
Authors: Peters J E , Bartoszyk I M , Dheer S , Benson S A
Published: November 2016

F plasmids use surface exclusion to prevent the redundant entry of additional F plasmids during active growth of the host cells. This mechanism is relaxed during stationary phase and nonlethal selections, allowing homosexual redundant plasmid transfer. Homosexual redundant transfer occurs in stationary-phase liquid cultures, within nongrowing populations on solid media, and on media lacking a carbon source. We examined the relationship between homosexual redundant transfer, which occurs between F+ hosts, and reversion of a plasmid-encoded lac mutant allele, lacI33omegalacZ. Sodium dodecyl sulfate (SDS) and mutations that prevent normal transfer to F- cells reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. A recA null mutation reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ mutation. Conversely, a recD null mutation increased redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. These results suggest an explanation for why SDS and these mutations affect reversion of the plasmid lacI33omegalacZ allele. However, a direct causal relationship between transfer and reversion remains to be established. These results suggest that Rec proteins play an active role in redundant transfer and/or that redundant transfer is regulated with the activation of recombination. Redundant homosexual plasmid transfer during a period of stress may represent a genetic response that facilitates evolution of plasmid-encoded functions through mutation, recombination, reassortment, and dissemination of genetic elements present in the populations.
A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus
Authors: Gilbert C , Atlan D , Blanc B , Portailer R , Germond J E , Lapierre L , Mollet B
Published: November 2016

Investigation of the chromosomal region downstream of the lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus revealed the presence of a gene (prtB) encoding a proteinase of 1,946 residues with a predicted molecular mass of 212 kDa. The deduced amino acid sequence showed that PrtB proteinase displays significant homology with the N termini and catalytic domains of lactococcal PrtP cell surface proteinases and is probably synthesized as a preproprotein. However, the presence of a cysteine near the histidine of the PrtB active site suggests that PrtB belongs to the subfamily of cysteine subtilisins. The C-terminal region strongly differs from those of PrtP proteinases by having a high lysine content, an imperfect duplication of 41 residues, and a degenerated sequence compared with the consensus sequence for proteins anchoring in the cell walls of gram-positive bacteria. Finally, the product of the truncated prtM-like gene located immediately upstream of the prtB gene seems too short to be involved in the maturation of PrtB.
A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA
Authors: Leibovitz E , Béguin P
Published: November 2016

The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.
Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination
Authors: Miesel L , Roth J R
Published: November 2016

A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay. The assay requires recombination within short (approximately 3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment. Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded. In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10- to 30-fold reduction. In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery. In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency. We suggest that these three proteins promote a rate-limiting step in the RecBC-dependent recombination process. The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions. When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.
The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface
Authors: Fernandez D , Dang T A , Spudich G M , Zhou X R , Berger B R , Christie P J
Published: November 2016

The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins. VirB7 was shown to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with [3H]palmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II. A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a delta virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function. VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a delta virB operon derivative transformed with a virB7 expression plasmid. Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E. coli and A. tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts. VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A. tumefaciens spheroplasts and remained intact upon treatment of whole cells. Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space. Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell.
Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2
Authors: Anthony K G , Kathir P , Moore D , Ippen-Ihler K , Frost L S
Published: November 2016

The sequence of a region of the F plasmid containing the traLEKBP genes involved in plasmid transfer was compared to the equivalent regions of two IncFII plasmids, R100-1 and ColB2. The traLEK gene products of all three plasmids were virtually identical, with the most changes occurring in TraE. The TraB genes were also nearly identical except for an 11-codon extension at the 3' end of the R100-1 traB gene. The TraP protein of R100-l differed from those of F and ColB2 at its N terminus, while the ColB2 TraP protein contained a change of sequence in a predicted loop which was shown to be exposed in the periplasmic space by TnphoA mutagenesis. The effect of the altered TraP sequences was determined by complementing a traP mutant with clones expressing the traKBP genes of F, R100-1, and ColB2. The traP mutation in pOX38 (pOX38-traP474), a derivative of F, was found to have little effect on pilus production, pilus retraction, and filamentous phage growth and only a moderate effect on transfer. The transfer ability of pOX38-traP474 was shown to be affected by mutations in the rfa (lipopolysaccharide) locus and in ompA in the recipient cell in a manner similar to that for the wild-type pOX38-Km plasmid itself and could be complemented with the traP analogs from R100-1 and ColB2 to give an F-like phenotype. Thus, the TraP protein appears to play a minor role in conjugation and may interact with TraB, which varies in sequence along with TraP, in order to stabilize the proposed transmembrane complex formed by the tra operon products.
Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis
Authors: Kwon O , Hudspeth M E , Meganathan R
Published: November 2016

In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2). Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene. Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen. An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy. Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically. An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media. These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene. Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.
Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism
Authors: Tong S , Porco A , Isturiz T , Conway T
Published: November 2016

Three genes involved in gluconate metabolism, gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, respectively, were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli,gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic analysis of GntU indicates an apparent Km for gluconate of 212 microM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are separated by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.
Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila
Authors: Alber B E , Ferry J G
Published: November 2016

The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.
The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome
Authors: Matsuura M , Noguchi T , Yamaguchi D , Aida T , Asayama M , Takahashi H , Shirai M
Published: November 2016

The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.