bluuebunny/crossref_metadata_2025 huggingface dataset:
Spring phenology dominates over shade in affecting seedling performance and plant attack during the growing season
Authors: McClory R. W. , van Dijk L. J. A. , Mutz J. , Ehrlén J. , Tack A. J. M.
Published: January 2020

Climate change is affecting both the abiotic environment and the seasonal timing of life history events, with potentially major consequences for plant performance and plant-associated food webs. Despite this, we lack insights into how effects of plant phenology on plant performance and food webs depend on environmental conditions, and to what extent effects of phenology and the environment on plant performance are direct vs. mediated by changes in the plant-associated community.

We conducted a multifactorial field experiment to test for the effect of spring phenology and shade onQuercus roburseedling traits and performance, as well as attacks by specialist plant pathogens, insects and small mammals.

Spring phenology strongly affected seedling performance whereas shade only affected leaf thickness and chlorophyll. Likewise, spring phenology strongly affected herbivore and pathogen attack, whereas shade and its interaction with spring phenology only explained a minor part of the variation. Small mammals preferentially attacked later phenology seedlings, which strongly affected plant survival, while insect herbivores and pathogens did not mediate the effect of spring phenology and shade on plant performance.

Synthesis:This study highlights that the effect of spring phenology outweighs the effect of environmental context on plant performance and plant attack during the growing season. Interestingly, small mammal herbivores, and not diseases and insect herbivores, may play a key role in mediating the effect of spring phenology on plant performance. Together, these findings advance our understanding of the consequences of climate-induced changes in spring phenology and the abiotic environment on plant performance within a community context.
ATP hydrolysis by the SNF2 domain of the ultraspecific maintenance methylase Dnmt5 drives recognition and modification of hemimethylated DNA
Authors: Dumesic Phillip A. , Stoddard Caitlin I. , Catania Sandra , Narlikar Geeta J. , Madhani Hiten D.
Published: January 2020

C. neoformansDnmt5 is an ultraspecific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step that is essential for DNA methylation. Such coupling occurs independently of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating the Dnmt5 ATP binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a ‘flipped-out’ conformation of the target cytosine bypass the SNF2 domain’s requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to base-flipping by the DNMT domain. These findings establish a new role for a SNF2 ATPase domain: controlling substrate recognition and catalysis by an adjoined enzymatic domain. This coupling may contribute to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.
Synthesis and crystal structure of a mixed alkaline-earth powellite, Ca0.84Sr0.16MoO4
Authors: Kissinger Ryan M. , Chong Saehwa , Riley Brian J. , Crum Jarrod V.
Published: January 2020

A mixed alkaline-earth powellite, Ca0.84Sr0.16MoO4(calcium strontium molybdate), was synthesized by a flux method and its crystal structure was solved using single-crystal X-ray diffraction (SC-XRD) data. The compound crystallized in theI41/aspace group as with a typical CaMoO4powellite, but with larger unit-cell parameters and unit-cell volume as a result of the partial incorporation of larger Sr cations into the Ca sites within the crystal. The unit cell and volume were well fitted with the trendline calculated from literature values, and the powder X-ray diffraction (P-XRD) pattern of the ground crystal is in good agreement with the calculated pattern from the solved structure.
Impact of loop diuretics on critically ill patients with a positive fluid balance
Authors: Libório A. B. , Barbosa M. L. , Sá V. B. , Leite T. T.
Published: January 2020

The impact of the use of loop diuretics to prevent cumulative fluid balance in non‐oliguric patients is uncertain. This is a retrospective study to estimate the association of time‐averaging loop diuretic exposure in a large population of non‐cardiac, critically ill patients with a positive fluid balance (> 5% of body weight). The exposure was loop diuretic and the main outcomes were 28‐day mortality, severe acute kidney injury and successful mechanical ventilation weaning. Time‐fixed and daily time‐varying variables were evaluated with a marginal structural Cox model, adjusting bias for time‐varying exposure and the presence of time‐dependent confounders. A total of 14,896 patients were included. Patients receiving loop diuretics had better survival (unadjusted hazard ratio 0.56, 95%CI 0.39–0.81 and baseline variables adjusted hazard ratio 0.53, 95%CI 0.45–0.62); after full adjusting, loop diuretics had no association with 28‐day mortality (full adjusted hazard ratio 1.07, 95%CI 0.74–1.54) or with reducing severe acute kidney injury occurrence during intensive care unit stay – hazard ratio 1.05 (95%CI 0.78–1.42). However, we identified an association with prolonged mechanical ventilation (hazard ratio 1.59, 95%CI 1.35–1.89). The main results were consistent in the sub‐group analysis for sepsis, oliguria and the study period (2002–2007 vs. 2008–2012). Also, equivalent doses of up to 80 mg per day of furosemide had no significant association with mortality. After adjusting for time‐varying variables, the time average of loop diuretic exposure in non‐cardiac, critically ill patients has no association with overall mortality or severe acute kidney injury; however, prolonged mechanical ventilation is a concern.
An unusual interatrial septum
Authors: Komorovsky Roman , Tran Huu Nghi
Published: January 2020

The images focus on a very rare atrial septal anatomy known as "double atrial septum with persistent interatrial space.” Only scarce reports of this anomaly are found in literature. Due to increasing use of catheter‐based interventions requiring a transseptal puncture, the recognition of this anomaly is essential for performing safe and effective procedures.
Rhythmic change of adipokinetic hormones diurnally regulates locust vitellogenesis and egg development
Authors: Zheng H. , Chen C. , Liu C. , Song Q. , Zhou S.
Published: January 2020

Adipokinetic hormones (AKHs), the neurohormones synthesized in the insect corpora cardiaca are known to mobilize lipids and carbohydrates for energy‐consuming activities including reproduction. However, both inhibitory and stimulatory effects of AKHs on insect reproduction have been reported, and the underlying mechanisms remain elusive. Using the migratory locust, Locusta migratoria, as a model system, we report here that AKHs are expressed in response to rhythmic diel change, and AKH III expression increases markedly at photophase. Diurnal injection of AKH III but not AKH I or AKH II in adult females stimulates vitellogenesis and egg development. In contrast, AKH treatment at scotophase represses female reproduction. RNA interference‐mediated knockdown of AKH receptor (AKHR) results in significantly reduced vitellogenin (Vg) expression in the fat body at photophase along with reduced Vg deposition in the ovary. AKHR knockdown also leads to decreased expression of Brummer, triacylglycerol lipase and trehalose transporter, accompanied by suppressed mobilization of triacylglycerol and trehalose. We propose that in addition to stimulating Vg expression at photophase, AKH/AKHR signalling is likely to regulate ovarian uptake of Vg via triacylglycerol mobilization and trehalose homeostasis. This study provides new insights into the understanding of AKH/AKHR signalling in the regulation of insect reproduction.
Authors’ reply to: Comment on the article by Dr. Toshima about alopecia areata multiplex following autologous dermal micrograft injection
Authors: Toshima S. , Kurihara Y. , Wang E.H.C. , Nomura H. , Hayashi Y. , Christiano A.M. , Amagai M. , Umegaki‐Arao N.
Published: January 2020

Linked article: L. Trovato, et al. J Eur Acad Dermatol Venereol 2020; 34: e219–e221. https://doi.org/10.1111/jdv.16176.
Cheese sauces: Characteristics of ingredients, manufacturing methods, microbiological and sensory aspects
Authors: Szafrańska Jagoda O. , Sołowiej Bartosz G.
Published: January 2020

During recent years, companies producing dairy products have observed an increase in the consumption of cheese sauces. The purpose of this review is to provide a critical overview of the most important properties of cheese sauces: ingredients, additives, processes, different methods used for their manufacturing, microbiological and sensory aspects, all of which is interesting both for the food industry and consumers. Because it is expected that the consumption of such products will increase, it is worth getting acquainted with current production processes for cheese sauces and the possibilities of modifying them in terms of consumer preferences, trends, and health‐promoting properties.

Practical applications

This review shows how the interactions among ingredients, the usage of hydrocolloids, emulsifying salts, and other functional components, and processing conditions can be handled to obtain cheese sauces with targeted characteristics. Moreover, advancements in recent years have enabled cheesemakers to increase the quality of cheese sauces in terms of nutritional value. These aspects could be interesting for the food industry (product development technologists), scientists, and consumers.
Reply to Comment on: The Clinical Course After Long‐Term Acute Care Hospital Admission Among Older Medicare Beneficiaries
Authors: Makam Anil N. , Nguyen Oanh Kieu , Halm Ethan A.
Published: January 2020

This reply comments on the letter by Amanda Dawson.
Pathways to Parenthood in Social and Family Contexts: Decade in Review, 2020
Authors: Guzzo Karen Benjamin , Hayford Sarah R.
Published: January 2020

Whether, and under what circumstances, people become parents has implications for individual identity, family relationships, the well‐being of adults and children, and population growth and age structure. Understanding the factors that influence pathways to parenthood is central to the study of families and can inform policies aimed at changing childbearing behaviors. This review summarizes recent trends in fertility as well as research on the predictors and correlates of childbearing, with a focus on the United States and on research most relevant to family scholars. U.S. fertility has declined in recent years, perhaps reflecting the influence of the Great Recession and increasing economic inequality, and there is growing divergence in behaviors across social class. Scholars of childbearing have developed theories to better understand how childbearing is shaped by life course processes and social contexts, but more work is needed to understand group differentials, refine measurement and theories, and better integrate men and couples. Childbearing research has important implications for social policy, but the application of research to policy is hindered by ideological factors.
A Socioecological Perspective on Intimate Partner Violence Research: A Decade in Review
Authors: Hardesty Jennifer L. , Ogolsky Brian G.
Published: January 2020

Intimate partner violence (IPV) is a significant public health issue impacting millions globally. To frame this decade in review, we organize the research published since 2010 at each of four ecological levels (individual, relational, community, and sociocultural) to demonstrate advances and gaps in each. At the individual and relational level, we review the prevalence, directionality, typologies, predictors, and outcomes of IPV. We attend to postseparation dynamics as well as research on youth exposure. We also discuss key theoretical advances. Our review of individual and relational research is more substantial as most research on IPV focuses on these factors with less attention to community and sociocultural contexts. Reflecting the state of the research within each ecological level, we review men's violence against women and incorporate developing research on men's victimization, reciprocal violence between men and women, and IPV among same‐sex partners. Throughout the review, we address key developments in knowledge as well as gaps and methodological strengths and limitations. We close with an integrated summary and recommendations for rigorous collaborative research across disciplines in the next decade to broaden our knowledge base and inform preventions and interventions.
Digging deeper? Biomass allocation patterns in trees and lianas in tropical seasonal forests
Authors: Lugli Laynara F.
Published: January 2020

This article is a Commentary on Smith‐Martin et al. 226: 714–726.
Controversial issues in soft tissue solitary fibrous tumors: A pathological and molecular review
Authors: Machado Isidro , Nieto‐Morales Gema , Cruz Julia , Navarro Samuel , Giner Francisco , Ferrandez Antonio , López‐Soto María Victoria , Lavernia Javier , Llombart‐Bosch Antonio
Published: January 2020

The clinical evolution of solitary fibrous tumor (SFT) remains unclear. Although various clinical, morphological and molecular criteria may indicate increased risk of malignancy, some SFT can still progress despite having a clearly benign appearance. Various risk stratification systems have been proposed, but unfortunately they are not sufficient to precisely determine the malignant potential. In this review, we discuss current knowledge on SFT, focusing on the following controversial issues: (i) the diverse morphologic spectrum: ‘the great simulator;’ (ii) malignant transformation or dedifferentiation; (iii) current risk stratification systems; and (iv) molecular factors associated with clinical evolution. The morphological spectrum of SFT and the list of differential diagnoses continue to expand. Both have increased considerably since the first descriptions of specific molecular alterations. A classification of malignant SFT should not be based on histology alone. The correlation of all pathological and molecular factors is recommended; its inclusion in risk stratification systems may help to improve diagnosis and prognosis.
Paraphyletic species no more – genomic data resolve a Pleistocene radiation and validate morphological species of the Melanoplus scudderi complex (Insecta: Orthoptera)
Authors: Huang Jen‐Pan , Hill JoVonn G. , Ortego Joaquín , Knowles L. Lacey
Published: January 2020

Rapid speciation events, with taxa generated over a short time period, are among the most investigated biological phenomena. However, molecular systematics often reveals contradictory results compared with morphological/phenotypical diagnoses of species under scenarios of recent and rapid diversification. In this study, we used molecular data from an average of over 29 000 loci per sample from RADseq to reconstruct the diversification history and delimit the species boundary in a short‐winged grasshopper species complex (Melanoplus scudderi group), where Pleistocene diversification has been hypothesized to generate more than 20 putative species with distinct male genitalic shapes. We found that, based on a maximum likelihood molecular phylogeny, each morphological species indeed forms a monophyletic group, contrary to the result from a previous mitochondrial DNA sequence study. By dating the diversification events, the species complex is estimated to have diversified during the Late Pleistocene, supporting the recent radiation hypothesis. Furthermore, coalescent‐based species delimitation analyses provide quantitative support for independent genetic lineages, which corresponds to the morphologically defined species. Our results also showed that male genitalic shape may not be predicted by evolutionary distance among species, not only indicating that this trait is labile, but also implying that selection may play a role in character divergence. Additionally, our findings suggest that the rapid speciation events in this flightless grasshopper complex might be primarily associated with the fragmentation of their grassland habitats during the Late Pleistocene. Collectively, our study highlights the importance of integrating multiple sources of information to delineate species, especially for a species complex that diversified rapidly, and whose divergence may be linked to ecological processes that create geographic isolation (i.e. fragmented habitats), as well as selection acting on characters with direct consequences for reproductive isolation (i.e. genitalic divergence).
A Parametric Investigation of Vane Showerhead Film Cooling by Pressure-Sensitive Paint Technique
Authors: Abdeh H. , Barigozzi G. , Ravelli S. , Rouina S.
Published: January 2020

In this study, a parametric analysis of the thermal performance of a nozzle vane cascade with a showerhead cooling system made of four rows of cylindrical holes was carried out by using the pressure-sensitive paint (PSP) technique. Coolant-to-mainstream blowing ratio (BR), density ratio (DR), main flow isentropic exit Mach number (Ma2is), and turbulence intensity level (Tu1) were the considered parameters. The cascade was tested in an atmospheric wind tunnel at Ma2is values ranging from 0.2 to 0.6, with an inlet turbulence intensity level of 1.6% and 9%, at variable injection conditions of BR = 2.0, 3.0, 4.0. Moreover, the influence of the DR on the leading-edge film-cooling performance was investigated: the testing was carried out at DR = 1.0, using nitrogen as foreign gas, and DR = 1.5, with carbon dioxide serving as a coolant. In the near-hole region, higher BR and Ma2is resulted in higher effectiveness, while higher mainstream turbulence intensity reduced the thermal coverage in between the rows of holes, whatever the BR is. Further downstream along the vane pressure side, the effectiveness was negatively affected by rising the BR but positively influenced by lowering the mainstream turbulence intensity. Moreover, a decrease in the DR caused a reduction in the film-cooling performance, whose extent depends on the injection condition.
An Inverse, Decision-Based Design Method for Robust Concept Exploration
Authors: Nellippallil Anand Balu , Mohan Pranav , Allen Janet K. , Mistree Farrokh
Published: January 2020

In this paper, we extend our previous work on a goal-oriented inverse design method to carry out inverse robust design by managing the uncertainty involved. The extension embodies the introduction of specific robust design goals and new robust solution constraints anchored in the mathematical constructs of Error Margin Indices (EMIs) and Design Capability Indices (DCIs) to determine “satisficing” robust design specifications across analytical model-based process chains. Contributions in this paper include the designer’s ability to explore satisficing robust solution regions when multiple conflicting goals and multiple sources of uncertainty are present. Using the goal-oriented inverse design method, robust solutions are propagated in an inverse manner. We demonstrate the efficacy of the method and the associated robust design functionalities using an industry-inspired hot rolling and cooling process chain example problem for the production of a steel rod. In this example, we showcase the formulation of multiple mechanical property goals for the end product using the robustness metrics and the exploration of satisficing robust solutions for material microstructure after the cooling process using the robust solution constraints. The robust solutions thus identified are communicated in an inverse manner using the design method to explore satisficing robust solutions for the microstructure generated after the hot rolling process. Using the example, we demonstrate the robust co-design of material, product, and associated manufacturing processes. The method and the associated design constructs are generic and support the formulation and inverse robust design exploration under uncertainty of similar problems involving a sequential, analytical model-based flow of information across process chains.
Use of filamentous cyanobacteria for biodegradation of organic pollutants
Authors: Kuritz T , Wolk C P
Published: January 2020

Biodegradation is increasingly being considered as a less expensive alternative to physical and chemical means of decomposing organic pollutants. Pathways of biodegradation have been characterized for a number of heterotrophic microorganisms, mostly soil isolates, some of which have been used for remediation of water. Because cyanobacteria are photoautotrophic and some can fix atmospheric nitrogen, their use for bioremediation of surface waters would circumvent the need to supply biodegradative heterotrophs with organic nutrients. This paper demonstrates that two filamentous cyanobacteria have a natural ability to degrade a highly chlorinated aliphatic pesticide, lindane (gamma-hexachlorocyclohexane); presents quantitative evidence that this ability can be enhanced by genetic engineering; and provides qualitative evidence that those two strains can be genetically engineered to degrade another chlorinated pollutant, 4-chlorobenzoate.
Bromate reduction by denitrifying bacteria
Authors: Hijnen W , Voogt R , Veenendaal H R , van der Jagt H , van der Kooij D
Published: January 2020

In the presence of bromide, ozonation as applied in water treatment results in the formation of bromate, an ion with carcinogenic properties. The reduction of bromate by mixed bacterial populations as well as pure cultures was studied under laboratory conditions. Bromate was reduced to bromide by a mixed bacterial population with and without a preceding nitrate reduction step in an anaerobically incubated medium with ethanol as the energy and carbon source at 20 and 25 deg C. The predominating bacteria isolated from the batches showing bromate reduction were identified as Pseudomonas spp. Strains of Pseudomonas fluorescens reduced BrO(inf3)(sup-) to Br(sup-) but at a much lower rate than the mixed bacterial population did. Nitrate is a preferred electron acceptor for the bromate-reducing bacteria. Bromate reduction did not occur in the presence of NO(inf3)(sup-), and the rate of bromate reduction was at least 100 times lower than the rate of nitrate reduction. Bromate was completely converted to Br(sup-), indicating that intermediates, e.g., BrO(inf2)(sup-), did not accumulate during bromate reduction.
Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens
Authors: Gerritsen L , van der Wolf J M , van Vuurde J , Ehlers R , Krasomil-Osterfel K C , Smits P H
Published: January 2020

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture.
Discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy and canonical variate analysis
Authors: Holt C , Hirst D , Sutherland A , MacDonald F
Published: January 2020

Infrared spectra of type cultures of the six recognized species of the genus Listeria and of Listeria grayi subsp. murrayi were recorded. By use of a library of 59 spectra, comprising at least six replicates of each type, discrimination by canonical variate analysis of the spectral amplitudes allowed all of the spectra to be correctly classified.
Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces
Authors: Eriksson P V , di Paola G N , Pasetti M F , Manghi M A
Published: January 2020

An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces. The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h. It could be used as a framework for a test system for quantifying P. fluorescens spoilage in meat products.
Methanogenic conversion of 3-s-methylmercaptopropionate to 3-mercaptopropionate
Authors: van der Maarel M , Jansen M , Hansen T A
Published: January 2020

Anaerobic metabolism of dimethylsulfoniopropionate, an osmolyte of marine algae, in anoxic intertidal sediments involves either cleavage to dimethylsulfide or demethylation to 3-S-methylmercaptopropionate (MMPA) and subsequently to 3-mercaptopropionate. The methanogenic archaea Methanosarcina sp. strain MTP4 (DSM 6636), Methanosarcina acetivorans DSM 2834, and Methanosarcina (Methanolobus) siciliae DSM 3028 were found to use MMPA as a growth substrate and to convert it stoichiometrically to 3-mercaptopropionate. Approximately 0.75 mol of methane was formed per mol of MMPA degraded; methanethiol was not detected as an intermediate. Eight other methanogenic strains did not carry out this conversion. We also studied the conversion of MMPA in anoxic marine sediment slurries. Addition of MMPA (500 (mu)M) resulted in the production of methanethiol which was subsequently converted to methane (417 (mu)M). In the presence of the antibiotics ampicillin, vancomycin, and kanamycin (20 (mu)g/ml each), 275 (mu)M methane was formed from 380 (mu)M MMPA; no methanethiol was formed during these incubations. Only methanethiol was formed from MMPA when 2-bromoethanesulfonate (25 mM) was added to a sediment suspension. These results indicate that in natural environments MMPA could be directly or indirectly a substrate for methanogenic archaea.
Viability of Vibrio vulnificus in Association with Hemocytes of the American Oyster (Crassostrea virginica)
Authors: Harris-Young L , Tamplin M L , Mason J W , Aldrich H C , Jackson J K
Published: January 2020

Certain indigenous estuarine bacteria, such as Vibrio vulnificus, may cause opportunistic human infections after consumption of raw oysters or exposure of tissues to seawater. V. vulnificus is known to be closely associated with oyster (Crassostrea virginica) tissues and is not removed by controlled purification methods, such as UV light-assisted depuration. In fact, when live shellfish are subjected to controlled purification, the number of V. vulnificus cells can markedly increase. A review of previous studies showed that few workers have examined mechanisms in oysters which may influence the persistence of V. vulnificus in shellfish, such as the fate of V. vulnificus following phagocytosis by molluscan hemocytes. The objectives of this study were to define the intracellular viability and extracellular viability of V. vulnificus during the phagocytic process and to study the release of specific lysosomal enzymes. The viability of a virulent estuarine V. vulnificus isolate with opaque morphology was compared with the viability of a translucent, nonvirulent form, the viability of Vibrio cholerae, and the viability of Escherichia coli in phagocytosis experiments. Our results showed that the levels of phagocytosis and bactericidal degradation of the opaque V. vulnificus isolate were less than the levels of phagocytosis and bactericial degradation of the translucent morphotype. These findings indicate that encapsulation may contribute to resistance to ingestion and degradation by hemocytes. The rates of intracellular death of V. cholerae and E. coli exceeded the rate of intracellular death of the opaque V. vulnificus isolate, even though the ingestion or uptake rates did not differ significantly. The levels of lysozyme activity and acid phosphatase activity were not significantly different in hemocyte monolayers inoculated with V. vulnificus.
Microbial Demethylation of Immunosuppressant FK-506: Isolation of 31-O-FK-506-Specific Demethylase Showing Cytochrome P-450 Characteristics from Streptomyces rimosus MA187
Authors: Shafiee A , Chen T , Cameron P
Published: January 2020

As a result of an extensive screening program for the microbial modification of the immunosuppressant FK-506, one culture, Streptomyces rimosus MA187, which specifically catalyzed the C-31 demethylation of FK-506 was identified. Treatment of the biotransforming culture with FK-506 increased demethylase activity 2.4-fold and stabilized the cytochrome P-450 protein. The enzyme responsible for this demethylation (31-O-FK-506 demethylase) was isolated and shown to be a soluble cytoplasmic protein which is constitutively expressed in the cells, which requires NADPH, ferredoxin-NADP(sup+)-reductase, and ferredoxin for activity, and which shows a cytochrome P-450 light absorption characteristic. Carbon monoxide saturation of the enzyme preparation and known mammalian cytochrome P-450 inhibitors such as quinidine HCl, ketoconazole, troleandomycin, and sulfaphenazole abolish the demethylating activity extensively. The purified enzyme is a monomeric protein with a molecular mass of 42 kDa and shows its maximal activity at a pH of 7.4 and an incubation temperature of 34(deg)C. The first 19 N-terminal amino acids in the sequence of the purified protein have been determined, with no cytochrome P-450 match found in the OWL and Swiss-Prot 23 databases. The isolated demethylase is therefore a cytochrome P-450 protein that can be used as a catalyst for the synthesis of 31-O-desmethylFK-506, an important immunosuppressant and a known metabolite of FK-506 metabolism by human liver microsomes.
stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans
Authors: Keller N P , Segner S , Bhatnagar D , Adams T H
Published: January 2020

Sterigmatocystin (ST) and aflatoxin are carcinogenic end point metabolites derived from the same biochemical pathway, which is found in several Aspergillus spp. Recently, an ST gene cluster, containing approximately 25 distinct genes that are each proposed to function specifically in ST biosynthesis, has been identified in Aspergillus nidulans. Each of these structural genes is named stc (sterigmatocystin) followed by a consecutive letter of the alphabet. We have previously described stcU (formerly verA) as encoding a keto-reductase required for the conversion of versicolorin A to ST. We now describe a second A. nidulans gene, stcS (formerly verB), that is located within 2 kb of stcU in the ST gene cluster. An stcS-disrupted strain of A. nidulans, TSS17, was unable to produce ST and converted ST/aflatoxin precursors to versicolorin A rather than ST, indicating that stcS functions at the same point in the pathway as stcU. Genomic sequence analysis of stcS shows that it encodes a cytochrome P-450 monooxygenase and constitutes a novel P-450 family, CYP59. Assuming that StcU activity mimics that of similar P-450s, it is likely that StcU catalyzes one of the proposed oxidation steps necessary to convert versicolorin A to ST. These results constitute the first genetic proof that the conversion of versicolorin A to ST requires more than one enzymatic activity.
Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530
Authors: B�ckle B , Galunsky B , M�ller R
Published: January 2020

A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.
Influence of Osmolarity on Phase Shift in Photorhabdus luminescens
Authors: Krasomil-Osterfel K C
Published: January 2020

The influence of osmolarity and other environmental factors like low oxygen levels, light, extreme pH values, and temperatures on phase variation of Photorhabdus luminescens, the symbiotic bacterium of entomopathogenic nematodes of the genus Heterorhabditis, was investigated. Only subculturing in low-osmolarity medium triggered a phase shift to secondary phase reliably.
Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis
Authors: Fleming A B , Tangney M , Jørgensen P L , Diderichsen B , Priest F G
Published: January 2020

A deletion of the spoIIAC gene of Bacillus licheniformis was prepared in vitro by using the splicing-by-overlap-extension technique. This gene was introduced into B. licheniformis on a temperature-sensitive plasmid, and following integration and excision from the chromosome, a precisely located deletion on the chromosomal gene was prepared. The mutated bacterium was totally asporogenous and formed abortively disporic cells characterized by asymmetric septa at the poles of the cells. Qualitative plate tests revealed that the bacterium synthesized normal levels of DNase, polygalacturonate lyase, protease, RNase, and xylanase, but the hydrolysis zones due to beta-1,3-glucanase and carboxymethyl cellulase activity were smaller in the mutant than in the parent strain. The synthesis of alkaline protease was the same in batch cultures of the mutant and the parent during prolonged incubation for 72 h, but the alpha-amylase yields were reduced by about 30% by the mutation.
Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica
Authors: Euverink G J , Hessels G I , Franke C , Dijkhuizen L
Published: January 2020

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)
Rhizobium tropici chromosomal citrate synthase gene
Authors: Hernández-Lucas I , Pardo M A , Segovia L , Miranda J , Martínez-Romero E
Published: January 2020

Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.
Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site
Authors: Boyce J D , Davidson B E , Hillier A J
Published: January 2020

Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp. cremoris H2. One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int). The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L. lactis strains.
Use of Starvation Promoters To Limit Growth and Selectively Enrich Expression of Trichloroethylene- and Phenol-Transforming Activity in Recombinant Escherichia coli
Authors: Matin A , Little C D , Fraley C D , Keyhan M
Published: January 2020

Volume 61, no. 9, p. 3323: the title of the article should read as shown above. [This corrects the article on p. 3323 in vol. 61.].
Uptake of Benzoic Acid and Chloro-Substituted Benzoic Acids by Alcaligenes denitrificans BRI 3010 and BRI 6011
Authors: Miguez C B , Greer C W , Ingram J M , Macleod R A
Published: January 2020

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.
A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination
Authors: Kiel J A , ten Berge A M , Borger P , Venema G
Published: January 2020

We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.
Genetic diversity of Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 in Kenya
Authors: Smith J J , Offord L C , Holderness M , Saddler G S
Published: January 2020

Genetic diversity among isolates of the bacterial plant pathogen Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 biovar II of Kenya was determined by PCR with repetitive sequences (ERIC and BOX repetitive primer sets) and pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE). The study comprised 46 isolates collected during 1992 from the major potato-growing regions of Kenya (45 were identified as race 3 biovar II, and 1 belonged to race 3 biovar N2) and 39 reference isolates from 19 other countries. RC-PFGE identified 10 distinct profile types among the Kenyan race 3 biovar II isolates (29 of the isolates exhibited identical profiles) and a further 27 distinct profile types among the reference isolates. ERIC and BOX primer sets were unable to differentiate race 3 biovar II isolates within the Kenyan population but differentiated a further two distinct profile types among the reference isolates. The race 3 biovar N2 isolate had a highly distinct RC-PFGE and repetitive sequence PCR profile. Statistical analysis of the data identified biogeographic trends consistent with conclusions drawn from previous studies on the origin and worldwide dissemination of race 3 biovar II isolates; however, genomic fingerprinting by RC-PFGE revealed a level of genetic diversity previously unrealized.
Direct detection of recombinant gene expression by two genetically engineered yeasts in soil on the transcriptional and translational levels
Authors: Tebbe C C , Wenderoth D F , Vahjen W , L�bke K , Munch J C
Published: January 2020

The expression of a recombinant gene by yeasts seeded into soil samples was directly measured by analyzing transcripts and gene product occurrences in soil extracts. Two yeast species, Saccharomyces cerevisiae WHL292 and Hansenula polymorpha LR9-Apr4, both engineered by a synthetic gene sequence encoding the mammalian peptide aprotinin, produced and secreted this peptide in batch cultures at concentrations of 90 and 64 ng ml-1, respectively. In S. cerevisiae, the aprotinin gene was located on plasmid p707 and expressed constitutively. H. polymorpha carried the gene chromosomally integrated, and its expression was inducible by methanol. To detect aprotinin transcripts, cells were directly lysed in the soil samples and the crude lysates were hybridized to oligo(dT)-coated magnetized polystyrene beads (Dynabeads). After separation and purification in a magnetic field, aprotinin mRNA was detected by reverse transcriptase PCR with aprotinin gene-specific primers. Transcripts from 10 cells g of soil-1 were sufficient for detection. When 10(7) cells of S. cerevisiae were inoculated into soil, aprotinin mRNA was detectable during the first 4 days. Addition of methanol and a combined nutrient solution was necessary to induce aprotinin gene expression of H. polymorpha in soil. Aprotinin could be detected directly in soil extracts by an indirect enzyme-linked immunosorbent assay with monoclonal aprotinin-specific antibodies. The detection threshold was 45 pg g of soil-1. In presterilized soil inoculated with S. cerevisiae (10(6) CFU g-1), aprotinin accumulated during the first 10 days to 12 ng g of soil-1 and then remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)
Comparison of the Growth and Survival of Larval Turbot in the Absence of Culturable Bacteria with Those in the Presence of Vibrio anguillarum, Vibrio alginolyticus, or a Marine Aeromonas sp
Authors: Munro P D , Barbour A , Birkbeck T H
Published: January 2020

Larval turbot (Scophthalmus maximus) were reared on rotifers (Brachionus plicatilis) in the absence of culturable bacteria for up to 14 days and exhibited growth and high rates of survival (>55% in five experiments). Low numbers of known bacteria were introduced into similar cultures by exposure of the rotifers to a suspension of bacteria prior to addition of rotifers to the larval cultures; Vibrio anguillarum 91079 caused a highly significant decrease (P <0.01) in the proportion of survivors in two separate trials. With an Aeromonas sp. previously isolated from a healthy batch of copepod-fed larvae, there was no significant difference in survival compared with control larvae, even though the density of bacteria in the water of larval cultures reached 10(sup7) ml(sup-1). Bacteria colonized the gut of larvae exposed to Aeromonas-treated rotifers to levels similar to those in conventionally reared fish (>4 x 10(sup4) CFU per larva). Rearing of larvae in the presence of known bacteria provides a means of investigating the interaction of specific bacteria with turbot larvae and could provide a method for the selection of bacteria which may restrict the growth of opportunistic pathogens which would be harmful to turbot larvae.
Arginine deiminase system and acid adaptation of oral streptococci
Authors: Curran T M , Lieou J , Marquis R E
Published: January 2020

Streptococcus rattus FA-1 and Streptococcus sanguis NCTC 10904 underwent phenotypic acid adaptation in batch cultures toward the end of sugar-fueled growth after the culture pH had dropped to triggering values. The bacteria could be derepressed or induced for arginine deiminase independently of acid adaptation, and arginolysis afforded protection against acid killing over and above that of acid adaptation.