bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Toughness Characteristics of API Grade Pipeline Steel Produced on Compact Strip Production (CSP) Line
Authors: Kendrick Vance , Frye Brian , Sutcliffe Alison , Rodriguez-Ibabe Jose M. , Stalheim Douglas G.
Published: November 2016

As concerns for environmental impact of oil and gas transmission pipelines and overall public safety of the transmission pipeline systems are raised in the public domain, development of optimum toughness characteristics are a key attribute. Toughness performance as measured by charpy impact testing or drop weight tear testing (DWTT) is heavily influenced first by the average transformed grain size and more importantly the cross sectional uniformity/distribution. In addition, the crystallographic texture created can further improve or detract from the toughness performance.

The final transformed cross sectional grain size along with the uniformity/distribution is heavily influenced by the available total metallurgical reduction ratio, microalloy design, proper generation of the various recrystallization behavior types during rolling, critical per pass reductions and the final post rolling cooling rate. The final crystallographic texture is influenced by final rolling temperature, cooling rate and final cooling stop temperature. When the final cross sectional grain size and overall uniformity/distribution are marginal for optimum toughness, the addition of favorable crystallographic textures can enhance the toughness performance.

The challenge of producing optimum toughness on a thin slab caster, <100 mm thickness, is well known due to the available metallurgical reduction ratios. Typically, for API grade steels, a metallurgical reduction ratio ≥7:1 is required in order to achieve optimum toughness. However, in a thin slab caster the maximum metallurgical reduction ratios possible can be between 5:1 and 7:1 depending on the final thickness.

Nucor Steel Gallatin has been working to optimize the overall toughness of API X-grades for transmission pipeline steels in thicknesses up to 12.7 mm using their thin slab Compact Strip Production (CSP) production facility. By utilizing a proper understanding of reducing the as-cast thin slab, along with the key alloy/process attributes and recrystallization behavior kinetics during the rolling process to optimize the final transformed cross sectional grain size and more importantly the uniformity/distribution, a high level of toughness performance can be realized. In addition, a further understanding of the contribution of specific crystallographic textures can further improve the toughness performance of these grades.

This paper will discuss alloy/process parameters that have been studied and optimized to improve the low temperature toughness of API steels. In addition, toughness performance and metallographic characterization of different processing parameters will be presented.
Cybersecurity From Field to Host
Authors: Cysouw Lee A. , Osburn Douglas C. , Rabadi Nader M.
Published: November 2016

Remote communications to field devices for data monitoring and controls has greatly reduced operating costs, reduced downtime, and helped to optimize our industry. With the ever growing threat of cyber-attacks, the need for securing that data is becoming a more common topic of discussion. Whether collecting SCADA or Measurement data from the field, doing remote configuration, or even sitting dormant, it is important to keep the line of communication to your devices secure. This presentation will discuss potential threats and examples of cyber-attacks. It will cover industry standards, types of cyber security, and the importance and best practices for securing data for Measurement and/or SCADA and control systems.
New Classification Approach for Dents With Metal Loss and Corrosion Along the Seam Weld
Authors: Nestleroth J. Bruce , Simek James , Ludlow Jed
Published: November 2016

The ability to characterize metal loss and gouging associated with dents and the identification of corrosion type near the longitudinal seam are two of the remaining obstacles with in-line inspection (ILI) integrity assessment of metal loss defects. The difficulty with denting is that secondary features of corrosion and gouging present very different safety and serviceability scenarios; corrosion in a dent is often not very severe while metal loss caused by gouging can be quite severe. Selective seam weld corrosion (SSWC) along older low frequency electric resistance welding (ERW) seams also presents two different integrity scenarios; the ILI tool must differentiate the more serious SSWC condition from the less severe conventional corrosion which just happens to be near a low frequency ERW seam. Both of these cases involve identification difficulties that require improved classification of the anomalies by ILI to enhance pipeline safety.

In this paper, two new classifiers are presented for magnetic flux leakage (MFL) tools since this rugged technology is commonly used by pipeline operators for integrity assessments. The new classifier that distinguishes dents with gouges from dents with corrosion or smooth dents uses a high and low magnetization level approach combined with a new method for analyzing the signals. In this classifier, detection of any gouge signal is paramount; the conservatism of the classifier ensures reliable identification of gouges can be achieved. In addition to the high and low field data, the classifier uses the number of distinct metal loss signatures at the dent, the estimated maximum metal loss depth, and the location of metal loss signatures relative to dent profile (e.g. Apex, Shoulder).

The new classifier that distinguishes SSWC from corrosion near the longitudinal weld uses two orientations of the magnetic field, the traditional axial field and a helical magnetic field. In this classifier, detection of any long narrow metal loss is paramount; the conservatism of the classifier ensures that high identification of SSWC can be achieved. The relative amplitude of the corrosion signal for the two magnetization directions is an important characteristic, along with length and width measures of the corrosion features.

These models were developed using ILI data from pipeline anomalies identified during actual inspections. Inspection measurements from excavations as well as pipe removed from service for lab analysis and pressure testing were used to confirm the results.
Pipeline Failures Resulting From Interacting Integrity Threats
Authors: Quickel Gregory T. , Beavers John A.
Published: November 2016

All pipelines contain manufacturing and construction anomalies that typically are stable with time and are not generally considered to be integrity threats. These include laminations, seam weld anomalies, girth weld anomalies, and shallow dents. There also are time-dependent integrity threats to buried natural gas and petroleum pipelines. These include external and internal corrosion, fatigue, corrosion fatigue, stress corrosion cracking (SCC), and hydrogen embrittlement. Unexpected failures can occur when the time dependent integrity threats are coupled with these stable anomalies. This paper describes several of these interactions.
Pipeline Rupture Detection Based on Machine Learning and Pattern Recognition
Authors: Di Blasi Martin , Li Zhan
Published: November 2016

Pipeline ruptures have the potential to cause significant economic and environmental impact in a short period of time, therefore it is critical for pipeline operators to be able to promptly detect and respond to them. Public stakeholder expectations are high and an evolving expectation is that the response to such events be automated by initiating an automatic pipeline shutdown upon receipt of rupture alarm. These types of performance expectations are challenging to achieve with conventional, model-based, leak-detection systems (i.e. CPM–RTTMs) as the reliability measured in terms of the false alarm rate is typically too low.

The company has actively participated on a pipeline-industry task force chaired by the API Cybernetics committee, focused on the development of best practices in the area of Rupture Recognition and Response. After API’s release of the first version of a Rupture Recognition and Response guidance document in 2014, the company has initiated development of its own internal Rupture Recognition Program (RRP). The RRP considers several rupture recognition approaches simultaneously, ranging from improvements to existing CPM leak detection to the development of new SCADA based rupture detection system (RDS). This paper will provide an overview of a specific approach to rupture detection based on the use of machine learning and pattern recognition techniques applied to SCADA data.
Making Integrity Decisions Using Metal Loss ILI Validation Process
Authors: Li Yanping , Fredine Gordon , Hubert Yvan , Hassanien Sherif
Published: November 2016

With the increased number of In-Line Inspections (ILI) on pipelines, it is important to evaluate ILI tool performance to support making rational integrity decisions. API 1163 “In-Line inspection systems qualification” outlines an ILI data set validation process which is mainly based on comparing ILI data with field measurements. The concept of comparing ILI results with previous ILI data is briefly mentioned in API 1163 Level 1 validation and discussed in detail in CEPA metal Loss ILI tool validation guidance document. However, a different approach from API 1163 is recommended in the CEPA document. Although the methodologies of validating an ILI performance are available, other than determining whether an inspection data set is acceptable, the role of ILI validation in integrity management decision making is not well defined in these documents.

Enbridge has reviewed API 1163 and CEPA methodologies and developed a process to validate metal loss ILI results. This process uses API 1163 as tool performance acceptance criteria while CEPA method is used to provide additional information such as depth over-call or under-call. The process captures the main concepts of both API 1163 and CEPA methodologies. It adds a new dimension to the validation procedure by evaluating different corrosion morphologies, depth ranges, and proximity to long seam and girth weld. The process also checks ILI results against previous ILI data sets and combines the results of several inspections. The validation results of one inspection provide information on whether the inspection data set is acceptable based on the ILI specification. This information is useful for excavation selection. Tool performance review based on several inspection data sets identifies the strength and weakness of an inspection tool; this information will be used to ensure the tool selection is appropriate for the expected feature types on the pipeline.

Applications of the validation process are provided to demonstrate how the process can aid in making integrity decisions and managing metal loss threats.
Minimum Counterbore Length and Taper Angle Criteria for Transition Welds
Authors: Dekhane Aditya , Liu Ming , Martens Michael
Published: November 2016

Transition welds joining pipes of unequal wall thickness are often used in gas and oil pipelines at road crossings, bends, fittings, and locations of class change. Pipeline operators have successively utilized a counterbore-tapered design for transition of unequal wall thickness. In the counterbore-tapered design, the wall thickness transition is moved away from a girth weld region, resulting in reduction of stress concentration near the girth weld, subsequently reducing the risk of hydrogen assisted cracking (HAC) and the driving force for fatigue crack growth.

The counterbore length (L) is an important design parameter in the design of the counterbore-tapered joint. The aim of specifying the minimum counterbore length is to ensure that any local moments caused by the interaction of longitudinal stress and the dissimilar wall thickness are satisfactorily attenuated when they reach the weld area. While determining the minimum counterbore length, the present counterbore-tapered design criteria considers the attenuation of the bending stress induced by the local moments but does not consider the bending stress magnitude. In certain cases, although the bending stress attenuation length is long, the magnitude of the bending stress is very small. The long counterbore length creates practical challenges for boring the pipe due to the limitation of boring tools. Since the magnitude of the bending stress is very small, it is not necessary to fully attenuate the bending stress. In this work, the minimum required counterbore length (L0) criteria was revised. The revised counterbore length criteria considered the magnitude of the bending stress and the stress attenuation, hence avoided the unnecessary long counterbore length. The revised criteria was based on an analytical solution and supported by detailed finite element analyses. The analyses showed that the minimum counterbore length can be greatly reduced for those cases required unnecessarily long counterbore length with negligible or small increase in the stress concentration in the weld area.

The present design method of counterbore-tapered joints allows only one value of taper angles at thickness transition, i.e., 14°. However, for some situations, creating the smoothness required with the 14° taper angle is difficult and an increase in the taper angle is preferred. In this work, it is shown that the stress concentration near the wall thickness transition and weld area for counterbore-tapered joints changes marginally by increasing taper angle from 14° to 30°.
Flexible mini gamma camera reconstructions of extended sources using step and shoot and list mode
Authors: Gardiazabal José , Matthies Philipp , Vogel Jakob , Frisch Benjamin , Navab Nassir , Ziegler Sibylle , Lasser Tobias
Published: November 2016

Purpose:

Hand‐ and robot‐guided mini gamma cameras have been introduced for the acquisition of single‐photon emission computed tomography (SPECT) images. Less cumbersome than whole‐body scanners, they allow for a fast acquisition of the radioactivity distribution, for example, to differentiate cancerous from hormonally hyperactive lesions inside the thyroid. This work compares acquisition protocols and reconstruction algorithms in an attempt to identify the most suitable approach for fast acquisition and efficient image reconstruction, suitable for localization of extended sources, such as lesions inside the thyroid.

Methods:

Our setup consists of a mini gamma camera with precise tracking information provided by a robotic arm, which also provides reproducible positioning for our experiments. Based on a realistic phantom of the thyroid including hot and cold nodules as well as background radioactivity, the authors compare “step and shoot” (SAS) and continuous data (CD) acquisition protocols in combination with two different statistical reconstruction methods: maximum‐likelihood expectation–maximization (ML‐EM) for time‐integrated count values and list‐mode expectation–maximization (LM‐EM) for individually detected gamma rays. In addition, the authors simulate lower uptake values by statistically subsampling the experimental data in order to study the behavior of their approach without changing other aspects of the acquired data.

Results:

All compared methods yield suitable results, resolving the hot nodules and the cold nodule from the background. However, the CD acquisition is twice as fast as the SAS acquisition, while yielding better coverage of the thyroid phantom, resulting in qualitatively more accurate reconstructions of the isthmus between the lobes. For CD acquisitions, the LM‐EM reconstruction method is preferable, as it yields comparable image quality to ML‐EM at significantly higher speeds, on average by an order of magnitude.

Conclusions:

This work identifies CD acquisition protocols combined with LM‐EM reconstruction as a prime candidate for the wider introduction of SPECT imaging with flexible mini gamma cameras in the clinical practice.
Calciprotein particles as potential etiologic agents of idiopathic preterm birth
Authors: Shook Lydia L. , Buhimschi Catalin S. , Dulay Antonette T. , McCarthy Megan E. , Hardy John T. , Duzyj Buniak Christina M. , Zhao Guomao , Buhimschi Irina A.
Published: November 2016

Aggregation of calciprotein particles in amniotic fluid may play a pathogenic role in idiopathic preterm birth.
Animal EnterotoxigenicEscherichia coli
Authors: Dubreuil J. Daniel , Isaacson Richard E. , Schifferli Dieter M.
Published: November 2016

EnterotoxigenicEscherichia coli(ETEC) is the most common cause ofE. colidiarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors: adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17, and F18 fimbriae. Once established in the animal small intestine, ETEC produce enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes: heat-labile toxins that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This review describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics, and the identification of potential new targets by genomics are presented in the context of animal ETEC.
Anaerobic Formate and Hydrogen Metabolism
Authors: Pinske Constanze , Sawers R. Gary
Published: November 2016

Numerous recent developments in the biochemistry, molecular biology, and physiology of formate and H
2
metabolism and of the [NiFe]-hydrogenase (Hyd) cofactor biosynthetic machinery are highlighted. Formate export and import by the aquaporin-like pentameric formate channel FocA is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate. Formate is disproportionated by the reversible formate hydrogenlyase (FHL) complex, which has been isolated, allowing biochemical dissection of evolutionary parallels with complex I of the respiratory chain. A recently identified sulfido-ligand attached to Mo in the active site of formate dehydrogenases led to the proposal of a modified catalytic mechanism. Structural analysis of the homologous, H
2
-oxidizing Hyd-1 and Hyd-5 identified a novel proximal [4Fe-3S] cluster in the small subunit involved in conferring oxygen tolerance to the enzymes. Synthesis of
Salmonella
Typhimurium Hyd-5 occurs aerobically, which is novel for an enterobacterial Hyd. The O
2
-sensitive Hyd-2 enzyme has been shown to be reversible: it presumably acts as a conformational proton pump in the H
2
-oxidizing mode and is capable of coupling reverse electron transport to drive H
2
release. The structural characterization of all the Hyp maturation proteins has given new impulse to studies on the biosynthesis of the Fe(CN)
2
CO moiety of the [NiFe] cofactor. It is synthesized on a Hyp-scaffold complex, mainly comprising HypC and HypD, before insertion into the apo-large subunit. Finally, clear evidence now exists indicating that
Escherichia coli
can mature Hyd enzymes differentially, depending on metal ion availability and the prevailing metabolic state. Notably, Hyd-3 of the FHL complex takes precedence over the H
2
-oxidizing enzymes.
Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli
Authors: Taverna P , Sedgwick B
Published: November 2016

Escherichia coli ada ogt mutants, which are totally deficient in O6-methylguanine-DNA methyltransferases, have an increased spontaneous mutation rate. This phenotype is particularly evident in starving cells and suggests the generation of an endogenous DNA alkylating agent under this growth condition. We have found that in wild-type cells, the level of the inducible Ada protein is 20-fold higher in stationary-phase and starving cells than in rapidly growing cells, thus enhancing the defense of these cells against DNA damage. The increased level of Ada in stationary cells is dependent on RpoS, a stationary-phase-specific sigma subunit of RNA polymerase. We have also identified a potential source of the mutagenic agent. Nitrosation of amides and related compounds can generate directly acting methylating agents and can be catalyzed by bacteria] enzymes. E. coli moa mutants, which are defective in the synthesis of a molybdopterin cofactor required by several reductases, are deficient in nitrosation activity. It is reported here that a moa mutant shows reduced generation of a mutagenic methylating agent from methylamine (or methylurea) and nitrite added to agar plates. Moreover, a moa mutation eliminates much of the spontaneous mutagenesis in ada ogt mutants. These observations indicate that the major endogenous mutagen is not S-adenosylmethionine but arises by bacterially catalyzed nitrosation.
Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes
Authors: Boch J , Kempf B , Schmid R , Bremer E
Published: November 2016

Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the choline-glycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline- or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to sigma A-dependent promoters and was stimulated by the presence of choline in the growth medium.
Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli
Authors: Ludwig A , Garcia F , Bauer S , Jarchau T , Benz R , Hoppe J , Goebel W
Published: November 2016

Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo. HlyA activated in E. coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity. Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site. The intact repeat domain of HlyA was not required for the activation. The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.
Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain wicky
Authors: Lunsford R D , London J
Published: November 2016

Streptococcus gordonii Wicky becomes competent only after stimulation with conditioned medium from strain Challis as a source of competence factor (CF). A 3.2-kbp genomic fragment from Challis was found to impart spontaneous competence on Wicky by a complementation assay. Wicky clones containing the fragment secreted a heat-sensitive activity that induced competence in Wicky and in a comA insertion mutant of Challis. Activity was localized to a putative open reading frame, comX, with the potential to encode a 52-amino-acid peptide. comX had no similarity to known sequences, and a comX::ermAM insertion mutant of Challis transformed normally and secreted CF. These data suggest that a CF-independent pathway for competence induction exists in S. gordonii.
Coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg belongs to the superfamily of adenylate-forming enzymes
Authors: Vermeij P , van der Steen R J , Keltjens J T , Vogels G D , Leisinger T
Published: November 2016

Depending on the reduction-oxidation state of the cell, some methanogenic bacteria synthesize or hydrolyze 8-hydroxyadenylylated coenzyme F420 (coenzyme F390). These two reactions are catalyzed by coenzyme F390 synthetase and hydrolase, respectively. To gain more insight into the mechanism of the former reaction, coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg was purified 89-fold from cell extract to a specific activity of 0.75 mumol.min-1.mg of protein-1. The monomeric enzyme consisted of a polypeptide with an apparent molecular mass of 41 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ftsA, the gene encoding coenzyme F390 synthetase, was cloned and sequenced. It encoded a protein of 377 amino acids with a predicted M(r) of 43,280. FtsA was found to be similar to domains found in the superfamily of peptide synthetases and adenylate-forming enzymes. FtsA was most similar to gramicidin S synthetase II (67% similarity in a 227-amino-acid region) and sigma-(L-alpha-aminoadipyl)-L-cysteine-D-valine synthetase (57% similarity in a 193-amino-acid region). Coenzyme F390 synthetase, however, holds an exceptional position in the superfamily of adenylate-forming enzymes in that it does not activate a carboxyl group of an amino or hydroxy acid but an aromatic hydroxyl group of coenzyme F420.
Isolation and characterization of a novel gamma-hexachlorocyclohexane-degrading bacterium
Authors: Thomas J C , Berger F , Jacquier M , Bernillon D , Baud-Grasset F , Truffaut N , Normand P , Vogel T M , Simonet P
Published: November 2016

The natural biotic capacity of soils to degrade gamma-hexachlorocyclohexane (gamma-HCH, lindane) was estimated using an enrichment technique based on the ability of soil bacteria to develop on synthetic media and degrade the xenobiotic compound, used as the sole source of carbon and energy. Bacterial inocula from relatively highly contaminated soils (from wood treatment factories) were found to promote efficiently the degradation of gamma-HCH, which subsequently permitted isolation of a competent gamma-HCH-degrading microorganism. The decrease of gamma-HCH concurrently with the release of chloride ions and the production of CO2 demonstrated the complete mineralization of gamma-HCH mediated by the isolate. This was confirmed by gas chromatography-mass spectrometry analyses showing that degradation subproducts of gamma-HCH included an unidentified tetrachlorinated compound and subsequently 1,2,4-trichlorobenzene and 2,5-dichlorophenol. The two linA- and linB-like genes coding, respectively, for a gamma-HCH dehydrochlorinase and a dehalogenase were characterized by using a PCR strategy based on sequence homologies with previously published sequences from Sphingomonas paucimobilis UT26. Nucleotide sequence analysis of the linA-like region revealed the presence of a 472-bp open reading frame exhibiting high homology with the linA gene from S. paucimobilis, while a preliminary study also indicated strong homology among the two linB genes. All enzymes involved in the gamma-HCH degradative pathway appear to be extracellular and encoded by genes located on the chromosome, although numerous cryptic plasmids have been detected.
Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone
Authors: Pozzi G , Masala L , Iannelli F , Manganelli R , Havarstein L S , Piccoli L , Simon D , Morrison D A
Published: November 2016

The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Havarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.
Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster
Authors: Simpson D A , Hammarton T C , Roberts I S
Published: November 2016

The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an E. coli sigma 70 promoter, with no similarities to binding sites for other sigma factors. Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site. In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter. The kpsF gene was 98.8% identical with the kpsF gene identified in the E. coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters. An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene. No essential role for KpsF in the expression of the K5 antigen could be established. The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C. Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression. Likewise, RfaH, which is known to regulate expression of E. coli group II capsules had no effect on the expression of region 1. Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.
Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region
Authors: Trinh S , Haggoud A , Reysset G
Published: November 2016

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.
Rapid identification and cloning of bacterial transferrin and lactoferrin receptor protein genes
Authors: Ogunnariwo J A , Schryvers A B
Published: November 2016

The sequences of genes encoding the transferrin and lactoferrin receptor proteins from several bacterial species were analyzed for areas of identity in the predicted protein sequences. Degenerate oligonucleotide primers were designed and tested for their ability to amplify portions of the receptor genes. Primer pairs capable of amplifying products of the tbpA/lbpA or tbpB/lbpB genes from all species possessing these receptors were identified.
Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease
Authors: Begley G S , Warner K A , Arents J C , Postma P W , Jacobson G R
Published: November 2016

We isolated 10 mannitol-positive mutants from a mannitol-negative Escherichia coli strain. These mutations mapped within ptsG, encoding the glucose permease (EIIGlc), and resulted in a G-320-to-V substitution that allows EIIGlc to transport mannitol. Gly-320 lies within a putative transmembrane helix of EIIGlc that may be involved in substrate recognition.
Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa
Authors: Luo Z , Sachs M S
Published: November 2016

The Neurospora crassa arg-2 transcript contains an upstream open reading frame (uORF) specifying a 24-residue leader peptide and is subject to a novel form of negative translational regulation in response to arginine. The role of the arg-2 uORF in arginine-specific negative regulation was investigated by using translational fusions of wild-type and mutant arg-2 sequences to the Escherichia coli lacZ reporter gene specifying beta-galactosidase. The wild-type uORF conferred Arg-specific regulation on the reporter gene in N. crassa, but mutated or truncated uORFs did not, as determined by measurements of beta-galactosidase activity produced in N. crassa strains expressing arg-2-lacZ fusion genes. All effects on reporter gene expression were posttranscriptional, as determined by measurement of RNA levels. Both sequence-dependent and sequence-independent effects of uORFs were observed. Genes containing the wild-type uORF or a 21-codon mutated uORF showed reduced translation in comparison with that of a gene lacking a uORF. Both uORF-containing transcripts showed reduced association with polysomes relative to transcripts lacking a uORF, but only the transcript with the wild-type uORF showed a reduced average number of ribosomes associated with it in response to arginine addition. Direct translational fusions between uORF sequences and lacZ sequences indicated that the uORF is translated. Overlapping the uORF with the lacZ initiation codon indicated that ribosome reinitiation at a downstream start codon is not integral to uORF-mediated, Arg-specific translational regulation. These studies provide direct biochemical evidence for arg-2 uORF function in translational control.
Purification of Vibrio cholerae fur and estimation of its intracellular abundance by antibody sandwich enzyme-linked immunosorbent assay
Authors: Watnick P I , Eto T , Takahashi H , Calderwood S B
Published: November 2016

The Vibrio cholerae fur gene was previously cloned and sequenced. A putative Fur box was identified in the divergent promoters of irgA, a virulence factor of V. cholerae, and irgB, a transcriptional activator of irgA. In this work, V. cholerae Fur was overexpressed in Escherichia coli and purified to approximately 95% homogeneity. The purified protein bound a DNA fragment containing the irgA-irgB promoter in a gel shift assay. The purified protein was used to raise monoclonal and polyclonal antibodies to V. cholerae Fur, and a Fur sandwich enzyme-linked immunosorbent assay was developed to estimate the intracellular abundance of Fur under a variety of growth conditions. The number of Fur molecules per cell during exponential growth was approximately 2,500, which is higher than most measurements for other bacterial repressors but comparable to the intracellular concentration of the leucine-responsive regulatory protein. The number of Fur molecules per cell increased in the late logarithmic and stationary phases. Growth of V. cholerae in low-iron medium did not alter the intracellular abundance of Fur significantly. Growth under microaerophilic conditions resulted in a significant, approximately twofold decrease in the intracellular levels of Fur. The measurements of intracellular Fur abundance indicate that a large amount of this repressor is produced constitutively and that the concentration of Fur in the cell varies by less than a factor of 2 under the conditions studied. We hypothesize that the high constitutive expression of Fur is necessary for its role as an iron-responsive regulator.
Nitrogen deprivation of Anabaena sp. strain PCC 7120 elicits rapid activation of a gene cluster that is essential for uptake and utilization of nitrate
Authors: Cai Y , Wolk C P
Published: November 2016

A transposon bearing luxAB, encoding luciferase, as a reporter of transcription was used to identify genes that are activated rapidly upon deprivation of Anabaena sp. strain PCC 7120 of fixed nitrogen. The three transposon-marked loci that were identified as responding most rapidly and strongly are closely linked and situated within nirA and nrtC and between nrtD and narB, genes whose products are responsible for uptake and reduction of NO2- and NO3-. A strain bearing a transcriptional fusion of narB to luxAB was constructed. Luminescence catalyzed by LuxAB was used to report on the expression of the interrupted genes. Whether these genes are regulated only coordinately is discussed.
Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli
Authors: Park H , Inouye M
Published: November 2016

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.
Evidence for two possible glnB-type genes in Herbaspirillum seropedicae
Authors: Benelli E M , Souza E M , Funayama S , Rigo L U , Pedrosa F O
Published: November 2016

Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate. One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms. A Tn5-20 mutation of this glnB was Nif negative.
Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT
Authors: Paterson E S , Iyer V N
Published: November 2016

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.
Regulation of yeast phospholipid biosynthetic genes in phosphatidylserine decarboxylase mutants
Authors: Griac P
Published: November 2016

In the yeast Saccharomyces cerevisiae, the products of two genes (PSD1 and PSD2) are able to catalyze the decarboxylation of phosphatidylserine (PS) to produce phosphatidylethanolamine (PE) (C. J. Clancey, S. Chang, and W. Dowhan, J. Biol. Chem. 268:24580-24590, 1993; P. J. Trotter, J. Pedretti, and D. R. Voelker, J. Biol. Chem. 268:21416-21424, 1993; P.J. Trotter, and D. R. Voelker, J. Biol. Chem. 270:6062-6070, 1995). I report that the major mitochondrial PS decarboxylase gene (PSD1) is transcriptionally regulated by inositol in a manner similar to that reported for other coregulated phospholipid biosynthetic genes. The second PS decarboxylase gene (PSD2) is not regulated on a transcriptional level by inositol and/or ethanolamine. In yeast, phosphatidylcholine (PC) biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. I show that the presence of a functional major mitochondrial PS decarboxylase encoded by the PSD1 gene is necessary for proper regulation of INO1 in response to inositol in the absence of ethanolamine. Disruption of the second PS decarboxylase gene (PSD2) does not affect the INO1 regulation. Analysis of phospholipid content of PS decarboxylase mutants suggests that the proportion of PC on total cellular phospholipids is not correlated to the cell's ability to repress INO1 in response to inositol. Rather, yeast cells are apparently able to monitor the flux through the phospholipid biosynthetic pathway and modify the transcription of phospholipid biosynthetic genes accordingly.
A possible role for acetylated intermediates in diaminopimelate and tabtoxinine-beta-lactam biosynthesis in Pseudomonas syringae pv. tabaci BR2.024
Authors: Liu L , Shaw P D
Published: November 2016

The deduced product of an open reading frame (ORF3) located in the tabtoxinine-beta-lactam (T beta L) biosynthetic region of Pseudomonas syringae pv. tabaci BR2.024 (BR2.024) has significant sequence homology to the dapD products of other bacteria. dapD encodes L-2,3,4,5-tetrahydrodipicolinate succinyl coenzyme A succinyltransferase (THDPA-ST), an enzyme in the diaminopimelate (DAP) and lysine biosynthetic pathway. Complementation studies, in vitro transcription-translation experiments, and enzymatic assays indicated that ORF3 encodes a product with THDPA-ST activity in Escherichia coli dapD mutant beta 274. However, a BR2.024 mutant with an insert in ORF3 was prototrophic, and only basal THDPA-ST activity was detected in extracts of both parent and mutant. This finding suggested that ORF3 was not required for DAP biosynthesis and that it did not encode a product with THDPA-ST activity. The results of enzymatic studies, indicating that BR2.024 uses acetylated intermediates for DAP biosynthesis, are consistent with the hypothesis that BR2.024 does not need THDPA-ST for DAP biosynthesis. The ORF3 mutant produced reduced levels of tabtoxin, indicating that ORF3 may have a role in T beta L biosynthesis. We have named the gene tabB and have proposed a possible function for the gene product.