bluuebunny/crossref_metadata_2025 huggingface dataset:
Longitudinal Stress Limits in Managing Spans and Other Longitudinal Loads
Authors: Wang Yong-Yi , Sen Millan , Song Peter
Published: November 2016

Stresses along the length of pipelines, termed longitudinal stresses, are generated by pipeline construction, service conditions, and changes in pipe support conditions. Although there are no explicit federal regulations limiting longitudinal stresses for in-service pipelines, pipeline operators typically reply on design requirements in established standards, such as ASME B31.4, B31.8, and CSA Z662, to manage possible integrity concerns arising from longitudinal stresses.

In this paper longitudinal stress limits in existing standards are reviewed. These standards include CSA Z662, ASME B31.4, ASME B31.8, DNV RP F101 and API RP 1111. These standards provide various formulae or specific values for the longitudinal stress limits. These limits are compared under various levels of internal pressure. Although the potential failure modes addressed by the different standards may be similar, the specific limits on longitudinal stresses differ among the standards. One of the interesting findings is that the limit on compressive longitudinal stress can be very low when the combined stress criteria (von Mises or Tresca) are applied to pipelines operating at a pressure level equivalent to Class 1 design of gas pipelines, i.e., hoop stress being 72% SMYS. The resulting compressive stress limits, at 18–29% SMYS by some standards, are much lower than often quoted 90%, 80%, or 54% SMYS limits. These quoted limits refer to limits for tensile stresses, but sometimes mistaken for compressive stress limits. The low compressive stress limits can be quite difficult to manage for spans after the addition of possible compressive stresses from temperature differential, lateral bending, and other sources.

Alternative stress limits that provide a consistent level of safety and in compliance with the spirits of standards are proposed. The new limits are sound from the viewpoint of safety, yet practical to apply. One possibility of increasing the compressive stress limit is reducing the hoop tensile stress by lowering operating pressure. The other possibility is setting a combined equivalent stress limit that is not overly conservative and preserves a sound level of safety. An Example is provided to illustrate the assessment of a span against the stress limits.
Effect of Residual Forming Stresses on Fracture in ERW Pipe
Authors: Anderson Ted L. , Brown Gregory W.
Published: November 2016

Many older pipelines contain significant residual stress due to the forming process. Cold expansion or a normalizing heat treatment can virtually eliminate residual forming stresses, but these practices were less common in the past. In the absence of cold expansion or normalization, residual forming stresses can be reduced by hydrostatic testing or operating pressures, but not eliminated entirely. Residual stresses can contribute to fracture in pipelines, particularly when the material toughness is low.

This article presents a series of analyses that seek to quantify the magnitude of residual forming stresses as well as their impact on pipeline integrity. The pipe forming process was simulated with elastic-plastic finite element analyses, which considered the effect of subsequent loading on relaxation of residual stresses. A second set of finite element simulations were used to quantify the effect of residual stresses on fracture behavior.
Simulation of Dynamic Crack Propagation and Arrest Using Various Types of Crack Arrestor
Authors: Uddin Mohammed , Wilkowski Gery
Published: November 2016

In linepipe steels, there has been a growing interest in using damage mechanics that provides physical models of the fracture process which are embedded into a two- or three-dimensional finite element (FE) model. Among the various damage models, the cohesive zone model (CZM) has recently been used to simulate the ductile crack growth behavior in linepipe steels because of its computational efficiency and it requires only two parameters which can be determined in experiments. While CZM is not yet to be used as predictive tool, but it has a great application in crack arrestor design as well as in providing insight to ductile crack propagation.

In this paper, the authors have demonstrated some practical applications of CZM in linepipe steels. The CZM was used to simulate the ductile crack propagation in full-scale pipes which was able to capture the global deformation as well as the experimental crack speed. The model was then used to determine the effect of anchor blocks at the end of the pipe in a large diameter full-scale burst test. Later, the model was used to simulate two small diameter pipe tests with steel crack arrestors to mimic two arrestor cases with one showing crack propagation and the other showing crack arrest. The CZM model was also applied to demonstrate the circumferential ring-off behavior of a small diameter pipe test with rigid crack arrestor. The arrestor model was then extended to simulate a large diameter full scale Mojave burst test with “soft crack arrestor (SCA)”. A single element FE model was developed to verify the SCA material which was later extended with stain-based failure criteria. Finally, ductile crack growth in full-scale pipe with SCA was demonstrated to show that the FE CZM model can be used to optimize the design of SCA.
Girth Welds Integrity Evaluation on a Vintage Gas Pipeline
Authors: Hryciuk Pedro M. , Minellono Jose A. , Domínguez Verónica
Published: November 2016

A leak on a girth weld was detected on a 609.6 mm (24 in) diameter transmission gas pipeline while recoating works were being performed. Failure analysis determined that the leak was caused by a pre-existing defect in the girth weld. This pre-existing defect had not been evolving over time. Nonetheless, because of the stress particular condition to which the gas pipeline was subject during the recoating work, the defect destabilized and progressed to failure.

In order to reduce the risk of occurrence of similar failures, a total of 27,000 girth welds were individually analyzed, corresponding to a length of 191 kilometers (118 miles) of the same gas pipeline section. The analysis included evaluation of internal inspection runs and girth welds gammagraphs available from the recoating work. Hence, not only was the data analyzed individually, but a comparison of the different sources of information to find a correlation was also performed.

Features detected in the analysis were classified and prioritized base on comparison with the failed girth weld defect. Also correspondence with crack like indications was considered. Severity of anomalies was categorized on four levels. A set of the highest severe crack like anomalies with potential failure were selected for direct assessment.

The complete process involved the assessment of 399 anomalies in total, revealing cracks on 79 girth welds. Metal reinforcements were installed as permanent repair on 27 girth welds according to repair criteria, and direct assessment findings were used to correlate internal inspection signals with features.

This paper discusses the results of the analysis, the field findings, and the actions taken.
A Study of Crack Interaction Criteria
Authors: Scott Colin
Published: November 2016

Cracks in close proximity may interact and lead to leaks or ruptures at pressures well below the predicted failure pressures of the individual cracks. Several industry organizations and standards, including CEPA, ASME, API, and British Standards provide guidance on the treatment of potentially interacting cracks. This guidance tends to be very conservative.

This paper is a study of crack interaction, including a discussion of industry guidance, a critical review of failure pressure models, and a review of results of laboratory hydro-testing of pipe sections containing either in-service flaws or simulated flaws. In some cases the industry guidance and current failure pressure models provide inconsistent predictions, and this leads to uncertainty in the assessments used in routine crack management programs. The results of the hydro-testing are discussed in the context of both types of predictions. Understanding and predicting these interactions is important in maintaining an effective and efficient crack management program.

The paper is aimed at engineers involved in integrity assessments and integrity management system process improvement.
Estimating J-R Curve From CVN Upper Shelf Energy and its Application
Authors: Tandon Samarth , Gao Ming , Krishnamurthy Ravi , Kania Richard , Rosca Gabriela
Published: November 2016

Accuracy in predictions of burst pressures for cracks in pipelines has significant impact on the pipeline integrity management decisions. One of the fracture mechanics models used for failure pressure prediction is API 579 Level 3 FAD ductile tearing instability analysis that requires J-R curves, i.e., crack resistance curves, for the assessment. However, J-R curves are usually unavailable for most pipelines. To overcome this technical barrier, efforts have been made to estimate the J-R curve indirectly from commonly available toughness data, such as the Charpy V-notched Impact Energy CVN values, by correlating the upper-shelf CVN value (energy) to the ductile fracture resistance J-R curve.

In this paper, the theoretical background and studies made by various researchers on this topic are reviewed. Attempts made by the present study to establish correlations between CVN and J-R curves for linepipe materials are then presented. Application of this CVN-JR correlation to API 579 Level 3 FAD tearing instability assessment for failure pressure predictions is demonstrated with examples. The accuracy of the correlation is analyzed and reported.
Microstructure and Toughness of Weld CGHAZ Under Different Heat Input for X90 High Strength Pipeline Steel
Authors: Yang Liuqing , Sui Yongli , Xia PeiPei , Yang Die , Zhang Yongqing
Published: November 2016

Two kinds of industry trial X90 pipeline steel which had different chemical composition were chosen as experimental materials, and the grain coarsening, microstructure evolution characteristics and the variation rules of low-temperature impact toughness in weld CGHAZ of this two steel under different welding heat input were studied by physical thermal simulation technology, SEM, optical microscope and Charpy impact test. The results show that microstructure in weld CGHAZ of 1# steel is mainly bainite ferrite (BF) and most of the M/A constituents are blocky or short rod-like; the grains of 2# steel are coarse and there is much granular bainite (GB), meanwhile M/A constituents become coarse and their morphology is changing from block to elongated laths; alloy content of X90 pipeline steel under different welding heat input has great effect on the grain size of original austenite, and when heat input is lower than 2.0KJ/mm, Charpy impact toughness in CGHAZ of lower alloy content pipeline steel is good; as heat input increases, impact toughness in CGHAZ of 1# steel is on the rise, and it is high (between 260J and 300J) when heat input is between 2.0KJ/mm and 2.5KJ/mm and the scatter of impact energy is small; impact toughness of 2# steel decreases gradually and the impact energy has obvious variability.
Leak Detection for Shut-In Pipelines
Authors: Vejahati Frank , Rostamy Noorallah , Noroozi Nader
Published: November 2016

In this paper, a new leak detection system based on a pattern recognition algorithm in a shut-in condition of a pipeline is presented. For a fully shut-in pipeline, the governing fluid dynamic equations are simplified to the thermodynamic state postulates. Hence, the shut-in section can be treated as a closed thermodynamic system with no mass flow in or out of the system boundaries. The system always contains the same amount of matter, but heat and work can be exchanged across the boundaries of the system. The pattern recognition algorithm presented in this paper automatically monitors the pressure drop patterns and generates an alarm when the pattern of pressure gradients matches the leak signatures. The algorithm takes into account the effect of thermal cooling and other operational complexities to enhance the reliability performance of the scheme. Results of the performance of the shut-in leak detection system are presented and discussed in this paper. Both simulated and historical leak scenarios during shut-in state are used to investigate the performance of the shut-in leak detection scheme.
Re-Routing of an Exposed Subsea Pipeline Using a Double Block and Bleed Intrusive Isolation Technique
Authors: Iyengar Srivatsan K. , Jaiswal Shivram
Published: November 2016

Oil & Natural Gas Corporation Ltd. (ONGC) operates a 42” gas pipeline that originates from an offshore platform and terminates at an onshore plant. The pipeline has operated for two decades and transmits 17% of India’s gas needs.

Unfortunately, the buried pipeline became exposed at Landfall Beach, likely due to the unpredictable impact of natural forces over a period of time. The shifting shoreline eroded resulting in the pipeline being exposed, which threatened its integrity. Stopgap arrangments were made to protect the pipeline from being hit and moved by continual tides, but that temporary measure also proved ineffective against the forces of the nature. The seasonal tropical storms worsened the situation.

ONGC rose to the challenge and decided to carry out permanent remedial measures, including re-routing of the affected segment of the pipeline and its associated station valve.

The situation presented several challenges. Because the pipeline was responsible for a large percentage of India’s gas needs, it was important to keep the pipeline live. Additionally, the worksite was a popular beach recreation area, so safety was a top priority.

ONGC contacted T.D. Williamson India Private Ltd. (TDW) for an isolation solution using double block and bleed methodogy to meet both of these concerns.
Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance
Authors: Welch Nicholas G. , Madiona Robert M. T. , Easton Christopher D. , Scoble Judith A. , Jones Robert T. , Muir Benjamin W. , Pigram Paul J.
Published: November 2016

Ensuring the optimum orientation, conformation, and density of substrate-bound antibodies is critical for the success of sandwich enzyme-linked immunosorbent assays (ELISAs). In this work, the authors utilize a diethylene glycol dimethyl ether plasma polymer (DGpp) coating, functionalized with chromium within a 96 well plate for the enhanced immobilization of a capture antibody. For an equivalent amount of bound antibody, a tenfold improvement in the ELISA signal intensity is obtained on the DGpp after incubation with chromium, indicative of improved orientation on this surface. Time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS) and principal component analysis were used to probe the molecular species at the surface and showed ion fragments related to lysine, methionine, histidine, and arginine coupled to chromium indicating candidate antibody binding sites. A combined x-ray photoelectron spectroscopy and ToF-SIMS analysis provided a surface molecular characterization that demonstrates antibody binding via the chromium complex. The DGpp+Cr surface treatment holds great promise for improving the efficacy of ELISAs.
In situ measurements of sediment acoustic properties in Currituck Sound and comparison to models
Authors: Lee Kevin M. , Ballard Megan S. , McNeese Andrew R. , Muir Thomas G. , Wilson Preston S. , Costley R. Daniel , Hathaway Kent K.
Published: November 2016

In situ measurements of compressional and shear wave speed and attenuation were collected 30 cm below the water-sediment interface in Currituck Sound, North Carolina at two field locations having distinctly different sediment types: medium-to-fine-grained sand and fine-grained sand with approximately 10% mud content. Shear wave measurements were performed with bimorph transducers to generate and receive horizontally polarized shear waves in the 300 Hz to 1 kHz band, and compressional wave measurements were performed using hydrophones operated in the 5 kHz to 100 kHz band. Sediment samples were collected at both measurement sites and later analyzed in the laboratory to characterize the sediment grain size distribution for each field location. Compressional and shear wave speed and attenuation were estimated from the acoustic measurements, and preliminary comparisons to the extended Biot model by Chotiros and Isakson [J. Acoust. Soc. 135, 3264–3279 (2014)] and the viscous grain-shearing theory by Buckingham [J. Acoust. Soc. 136, 2478–2488 (2014)] were performed.
Weight Regain: No Link to Success in a Real-Life Multiday Boxing Tournament
Authors: Reale Reid , Cox Gregory R. , Slater Gary , Burke Louise M.
Published: November 2016

Purpose:

Combat-sport athletes acutely reduce body mass (BM) before weigh-in in an attempt to gain a size/strength advantage over smaller opponents. Few studies have investigated these practices among boxers and none have explored the impact of this practice on competitive success.

Methods:

One hundred (30 women, 70 men) elite boxers participating in the Australian national championships were weighed at the official weigh-in and 1 h before each competition bout. Regain in BM after weigh-in was compared between finalists and nonfinalists, winners and losers of each fight, men and women, and weight divisions. Boxers were surveyed on their pre- and post-weigh-in nutrition practices.

Results:

The lightest men’s weight category displayed significantly greater relative BM regain than all other divisions, with no difference between other divisions. BM prebout was higher than official weigh-in for men (2.12% ± 1.62%; P < .001; ES = 0.13) and women (1.49% ± 1.65%; P < .001; ES = 0.11). No differences in BM regain were found between finalists and nonfinalists, winners and losers of individual bouts, or between preliminary or final bouts. BM regain was significantly greater (0.37% BM, P < .001; ES = 0.25) before an afternoon bout compared with a morning bout.

Conclusions:

Boxers engage in acute BM-loss practices before the official competition weigh-in, but this does not appear to affect competition outcomes, at least when weight regain between weigh-in and fighting is used as a proxy for the magnitude of acute loss. While boxers recognize the importance of recovering after weigh-in, current practice is not aligned with best-practice guidance.
Wearable Training-Monitoring Technology: Applications, Challenges, and Opportunities
Authors: Cardinale Marco , Varley Matthew C.
Published: November 2016

The need to quantify aspects of training to improve training prescription has been the holy grail of sport scientists and coaches for many years. Recently, there has been an increase in scientific interest, possibly due to technological advancements and better equipment to quantify training activities. Over the last few years there has been an increase in the number of studies assessing training load in various athletic cohorts with a bias toward subjective reports and/or quantifications of external load. There is an evident lack of extensive longitudinal studies employing objective internal-load measurements, possibly due to the cost-effectiveness and invasiveness of measures necessary to quantify objective internal loads. Advances in technology might help in developing better wearable tools able to ease the difficulties and costs associated with conducting longitudinal observational studies in athletic cohorts and possibly provide better information on the biological implications of specific external-load patterns. Considering the recent technological developments for monitoring training load and the extensive use of various tools for research and applied work, the aim of this work was to review applications, challenges, and opportunities of various wearable technologies.
The Live Attenuated Cholera Vaccine CVD 103-HgR Primes Responses to the Toxin-Coregulated Pilus Antigen TcpA in Subjects Challenged with Wild-Type Vibrio cholerae
Authors: Mayo-Smith Leslie M. , Simon Jakub K. , Chen Wilbur H. , Haney Douglas , Lock Michael , Lyon Caroline E. , Calderwood Stephen B. , Kirkpatrick Beth D. , Cohen Mitchell , Levine Myron M. , Gurwith Marc , Harris Jason B.
Published: November 2016

One potential advantage of live attenuated bacterial vaccines is the ability to stimulate responses to antigens which are only expressed during the course of infection. To determine whether the live attenuated cholera vaccine CVD 103-HgR (Vaxchora) results in antibody responses to the
in vivo
-induced toxin-coregulated pilus antigen TcpA, we measured IgA and IgG responses to
Vibrio cholerae
O1 El Tor TcpA in a subset of participants in a recently reported experimental challenge study. Participants were challenged with
V. cholerae
O1 El Tor Inaba N16961 either 10 days or 90 days after receiving the vaccine or a placebo. Neither vaccination nor experimental infection with
V. cholerae
alone resulted in a robust TcpA IgG or IgA response, but each did elicit a strong response to cholera toxin. However, compared to placebo recipients, vaccinees had a marked increase in IgG TcpA antibodies following the 90-day challenge, suggesting that vaccination with CVD 103-HgR resulted in priming for a subsequent response to TcpA. No such difference between vaccine and placebo recipients was observed for volunteers challenged 10 days after vaccination, indicating that this was insufficient time for vaccine-induced priming of the TcpA response. The priming of the response to TcpA and potentially other antigens expressed
in vivo
by attenuated
V. cholerae
may have relevance to the maintenance of immunity in areas where cholera is endemic.
Identification and characterization of BpH2, a novel histone H1 homolog in Bordetella pertussis
Authors: Goyard S
Published: November 2016

A basic protein, BpH2, with an apparent molecular mass of 18 kDa was purified from Bordetella pertussis, and the corresponding gene, bph2, was cloned. Sequence analysis revealed some homology to the H1 class of eukaryotic histones and to AlgP protein of Pseudomonas aeruginosa. BpH2 binds both single- and double-stranded DNA in a nonspecific manner. Deletion of the corresponding gene in B. pertussis generated a BpH2 null mutant with an altered growth rate in which the expression of two virulence factors, adenylate cyclase-hemolysin (CyaA) and filamentous hemagglutinin (FhaB), was reduced. It is suggested that BpH2 may exhibit specific regulatory functions through its interaction with chromosomal DNA.
The proton motive force generated in Leuconostoc oenos by L-malate fermentation
Authors: Salema M , Lolkema J S , San Romão M V , Lourero Dias M C
Published: November 2016

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.
mioC transcription, initiation of replication, and the eclipse in Escherichia coli
Authors: Bogan J A , Helmstetter C E
Published: November 2016

The potential role of mioC transcription as a negative regulator of initiation of chromosome replication in Escherichia coli was evaluated. When initiation was aligned by a shift of dnaC2(Ts) mutants to nonpermissive temperature (40 degrees C), mioC transcript levels measured at the 5' end or reading through oriC disappeared within one mass doubling. Upon return to permissive temperature (30 degrees C), the transcripts reappeared coordinately about 15 min after the first synchronized initiation and then declined sharply again 10 min later, just before the second initiation. Although these observations were consistent with the idea that mioC transcription might have to be terminated prior to initiation, it was found that the interval between initiations at permissive temperature, i.e., the eclipse period, was not influenced by the time required to shut down mioC transcription, since the eclipse was the same for chromosomes and minichromosomes which lacked mioC transcription. This finding did not, in itself, rule out the possibility that mioC transcription must be terminated prior to initiation of replication, since it might normally be shut off before initiation, and never be limiting, even during the eclipse. Therefore, experiments were performed to determine whether the continued presence of mioC transcription during the process of initiation altered the timing of initiation. It was found that minichromosomes possessing a deletion in the DnaA box upstream of the promoter transcribed mioC continuously and replicated with the same timing as those that either shut down expression prior to initiation or lacked expression entirely. It was further shown that mioC transcription was present throughout the induction of initiation by addition of chloramphenicol to a dnaA5(Ts) mutant growing at a semipermissive temperature. Thus, transcription through oriC emanating from the mioC gene promoter is normally inhibited prior to initiation of replication by the binding of DnaA protein, but replication can initiate with the proper timing even when transcription is not shut down; i.e., mioC does not serve as a negative regulator of initiation. It is proposed, however, that the reappearance and subsequent disappearance of mioC transcription during a 10-min interval at the end of the eclipse serves as an index of the minimum time required for the establishment of active protein-DNA complexes at the DnaA boxes in the fully methylated origin region of the chromosome. On this basis, the eclipse constitutes the time for methylation of the newly formed DNA strands (15 to 20 min at 30 degrees C) followed by the time for DnaA protein to bind and activate oriC for replication (10 min).
Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6
Authors: Yoon K S , Ishii M , Igarashi Y , Kodama T
Published: November 2016

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.
Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5
Authors: Dickens M L , Ye J , Strohl W R
Published: November 2016

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located just upstream of the daunomycin polyketide biosynthesis genes, revealed the presence of six complete genes. The two genes reading right to left include genes encoding the potentially translationally coupled gene products, an acyl carrier protein and a ketoreductase, and the four genes reading divergently, left to right, include two open reading frames of unknown function followed by a gene encoding an apparent glycosyltransferase and dauE, encoding aklaviketone reductase. Extracts of Streptomyces lividans TK24 containing recombinant DauE catalyzed the NADPH-specific conversion of aklaviketone, maggiemycin, and 7-oxodaunomycinone to aklavinone, epsilon-rhodomycinone, and daunomycinone, respectively. Neither the product of dauB nor that of the ketoreductase gene directly downstream of the acyl carrier protein gene demonstrated aklaviketone reductase activity.
ADP-ribosylation of proteins in Bacillus subtilis and its possible importance in sporulation
Authors: Huh J W , Shima J , Ochi K
Published: November 2016

Endogenous ADP-ribosylation was detected in Bacillus subtilis, as determined in vitro with crude cellular extracts. The ADP-ribosylated protein profile changed during growth in sporulation medium, displaying a temporary appearance of two ADP-ribosylated proteins (36 and 58 kDa) shortly after the end of exponential growth. Mutants resistant to 3-methoxybenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained, and a significant proportion (15%) were found to be defective in both sporulation and antibiotic production. These mutants failed to ADP-ribosylate the 36- and 58-kDa proteins. The parent strain also lost the ability to ADP-ribosylate these proteins when grown in the presence of 3-methoxybenzamide at a concentration at which sporulation but not cell growth was severely inhibited. Results from genetic transformations showed that the mutation conferring resistance to 3-methoxybenzamide, named brgA, was cotransformed with the altered phenotypes, i.e., defects in ADP-ribosylation and sporulation. spoOA and spoOF mutants displayed an ADP-ribosylation profile similar to that of the parent strain, but a spoOH mutant failed to ADP-ribosylate any proteins, including the 36- and 58-kDa proteins. The significance of protein ADP-ribosylation in sporulation was further indicated by the observation that ADP-ribosylation of the 36-kDa protein could be induced by treatment with decoyinine, an inhibitor of GMP-synthetase, and by amino acid limitation, both of which resulted in an immediate decrease in GTP pool size eventually leading to massive sporulation. We propose that a new sporulation gene, which presumably controls sporulation via ADP-ribosylation of certain functional proteins, exists.
Analysis of Fur binding to operator sequences within the Neisseria gonorrhoeae fbpA promoter
Authors: Desai P J , Angerer A , Genco C A
Published: November 2016

The gene encoding Neisseria gonorrhoeae periplasmic binding protein FbpA contains two regions whose sequences exhibit homology with the Escherichia coli ferric uptake regulator protein (Fur) consensus binding sequence. In this study, DNase I footprinting experiments were employed to characterize the operator sequences within the fbpA promoter region to which E. coli Fur binds. A 160-bp fragment encompassing the promotor region and the putative iron boxes of the fbpA promoter was incubated with Fur, DNaseI was added, and the products of these reactions were sequenced to identify nucleotide peaks that were protected. At 50 nM Fur, a protected region that spanned 33 bp and extended 19 bp upstream and 8 bp downstream of the -35 region of the fbpA promoter was observed. At higher concentrations of Fur (75 and 100 nM), an extension of this protected region upstream of the -35 region was observed. Introduction of a plasmid carrying an fbpA-cat transcriptional fusion in E. coli H1717 (Fur+) resulted in an 88% induction of chloramphenicol acetyltransferase expression under conditions of iron restriction; however, chloramphenicol acetyltransferase expression was not responsive to iron in E. coli H1745 (Fur-), indicating that transcriptional regulation of fbpA in response to iron occurs via the negative regulator Fur. The extent of the fbpA operator sequence (42 bp), as defined by our footprinting analysis, would suggest the binding of two Fur repressor dimers.
Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac
Authors: Conter A , Bouché J P , Dassain M
Published: November 2016

A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.
Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine
Authors: Peter H , Burkovski A , Krämer R
Published: November 2016

Corynebacterium glutamicum accumulates glycine betaine under conditions of high osmolarity. Previous work revealed the existence of a high-affinity glycine betaine permease which is osmotically regulated. In the present study, the corresponding gene was cloned. The betP gene, encoding the glycine betaine uptake carrier, was isolated by heterologous complementation of mutant strain Escherichia coli MKH13. From sequence analysis it is predicted to encode a protein of 595 amino acids. This protein shares 36% identity with the choline transport system BetT and 28% identity with the carnitine transport system CaiT of E. coli, as well as 38% identity with a protein with an unknown function from Haemophilus influenzae. Analysis of hydropathy indicated a common structure for all four transport proteins. After heterologous expression of betP in E. coli MKH13, the measured Km values for glycine betaine and the cotransported Na+ were similar to those found in C. glutamicum, whereas the modulation of activity by osmotic gradients was shifted to lower osmotic values.
The Bradyrhizobium japonicum rpoH1 gene encoding a sigma 32-like protein is part of a unique heat shock gene cluster together with groESL1 and three small heat shock genes
Authors: Narberhaus F , Weiglhofer W , Fischer H M , Hennecke H
Published: November 2016

The heat shock response of Bradyrhizobium japonicum is controlled by a complex network involving two known regulatory systems. While some heat shock genes are controlled by a highly conserved inverted-repeat structure (CIRCE), others depend on a sigma 32-type heat shock sigma factor. Using Western blot (immunoblot) analysis, we confirmed the presence of a sigma 32-like protein in B. japonicum and defined its induction pattern after heat shock. A B. japonicum rpoH-like gene (rpoH1) was cloned by complementation of an Escherichia coli strain lacking sigma 32. A knockout mutation in rpoH1 did not abolish sigma 32 production in B. japonicum, and the rpoH1 mutant showed the wild-type growth phenotype, suggesting the presence of multiple rpoH homologs in this bacterium. Further characterization of the rpoH1 gene region revealed that the rpoH1 gene is located in a heat shock gene cluster together with the previously characterized groESL1 operon and three genes encoding small heat shock proteins in the following arrangement: groES1, groEL1, hspA, rpoH1, hspB, and hspC. Three heat-inducible promoters are responsible for transcription of the six genes as three bicistronic operons. A sigma 32-dependent promoter has previously been described upstream of the groESL1 operon. Although the hspA-rpoH1 and hspBC operons were clearly heat inducible, they were preceded by sigma 70-like promoters. Interestingly, a stretch of about 100 bp between the transcription start site and the start codon of the first gene in each of these two operons was nearly identical, making it a candidate for a regulatory element potentially allowing heat shock induction of sigma 70-dependent promoters.
Adhesion of the positively charged bacterium Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon
Authors: Jucker B A , Harms H , Zehnder A J
Published: November 2016

Medical implants are often colonized by bacteria which may cause severe infections. The initial step in the colonization, the adhesion of bacteria to the artificial solid surface, is governed mainly by long-range van der Waals and electrostatic interactions between the solid surface and the bacterial cell. While van der Waals forces are generally attractive, the usually negative charge of bacteria and solid surfaces leads to electrostatic repulsion. We report here on the adhesion of a clinical isolate, Stenotrophomonas maltophilia 70401, which is, at physiological pH, positively charged. S. maltophilia has an electrophoretic mobility of +0.3 x 10(-8) m2 V-1 s-1 at pH 7 and an overall surface isoelectric point at pH 11. The positive charge probably originates from proteins located in the outer membrane. For this bacterium, both long-range forces involved in adhesion are attractive. Consequently, adhesion of S. maltophilia to negatively charged surfaces such as glass and Teflon is much favored compared with the negatively charged bacterium Pseudomonas putida mt2. While adhesion of negatively charged bacteria is impeded in media of low ionic strength because of a thick negatively charged diffuse layer, adhesion of S. maltophilia was particularly favored in dilute medium. The adhesion efficiencies of S. maltophilia at various ionic strengths could be explained in terms of calculated long-range interaction energies between S. maltophilia and glass or Teflon.
Polynucleotide phosphorylase of Escherichia coli is required for the establishment of bacteriophage P4 immunity
Authors: Piazza F , Zappone M , Sana M , Briani F , Dehò G
Published: November 2016

Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls. It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination. To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization. One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA. By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3'-to-5' exonucleolytic enzyme. A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1. These results indicate that the polynucleotide phosphorylase of E. coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.
Kinetics of expression of the Escherichia coli cad operon as a function of pH and lysine
Authors: Neely M N , Olson E R
Published: November 2016

The Escherichia coli cadBA genes are regulated at the transcriptional level by external pH and lysine. The membrane-localized CadC protein is required for activation of this operon under inducing conditions, which include acidic external pH, lysine, and oxygen limitation. To better understand the mechanism by which CadC functions, the kinetics of cadBA expression as a function of pH and lysine were examined. By primer extension assays, cadBA expression was detected within 4 min following exposure of cells to one of the inducing stimuli (low pH or lysine), provided that the cells had first been grown to steady state in the presence of the other inducing stimulus. The induction time was three to four times longer when both inducing stimuli were added simultaneously. cadBA expression was shut off within 4 min following a shift from acidic to neutral pH. Treatment of cells with chloramphenicol prevented induction by acidic pH and lysine. Transcription of lysP (encodes a lysine transporter) was also examined, since it is a negative regulator of cadBA expression in the absence of lysine. lysP expression was repressed by lysine but not influenced by pH. Putative transcription start sites for lysP and cadC were determined. Together, these data suggest that CadC senses the lysine- and pH-induced signals separately and that one of the roles of lysine in inducing cadBA may be to repress expression of lysP, thus eliminating the repressing effects of LysP.
Physical and genetic chromosomal map of an M type 1 strain of Streptococcus pyogenes
Authors: Suvorov A N , Ferretti J J
Published: November 2016

A physical map of the chromosome of an M type 1 strain of Streptococcus pyogenes was constructed following digestion with three different restriction enzymes, SmaI, SfiI, and SgrAI, and separation and analysis of fragments by pulsed-field gel electrophoresis. The genome size of this strain was estimated to be 1,920 kb. By employing Southern hybridization and PCR analysis, 36 genes were located on the map.
Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box
Authors: Marconi R T , Sung S Y , Hughes C A , Carlyon J A
Published: November 2016

In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.
Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis
Authors: Nübel U , Engelen B , Felske A , Snaidr J , Wieshuber A , Amann R I , Ludwig W , Backhaus H
Published: November 2016

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.