bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic signatures of geographic isolation and natural selection in coral reef fishes
Authors: Gaither Michelle R. , Bernal Moisés A. , Coleman Richard R. , Bowen Brian W. , Jones Shelley A. , Simison W. Brian , Rocha Luiz A.
Published: March 2015

The drivers of speciation remain among the most controversial topics in evolutionary biology. Initially, Darwin emphasized natural selection as a primary mechanism of speciation, but the architects of the modern synthesis largely abandoned that view in favour of divergence by geographic isolation. The balance between selection and isolation is still at the forefront of the evolutionary debate, especially for the world's tropical oceans where biodiversity is high, but isolating barriers are few. Here, we identify the drivers of speciation in Pacific reef fishes of the genus Acanthurus by comparative genome scans of two peripheral populations that split from a large Central‐West Pacific lineage at roughly the same time. Mitochondrial sequences indicate that populations in the Hawaiian Archipelago and the Marquesas Islands became isolated approximately 0.5 Ma. The Hawaiian lineage is morphologically indistinguishable from the widespread Pacific form, but the Marquesan form is recognized as a distinct species that occupies an unusual tropical ecosystem characterized by upwelling, turbidity, temperature fluctuations, algal blooms and little coral cover. An analysis of 3737 SNPs reveals a strong signal of selection at the Marquesas, with 59 loci under disruptive selection including an opsin Rh2 locus. While both the Hawaiian and Marquesan populations indicate signals of drift, the former shows a weak signal of selection that is comparable with populations in the Central‐West Pacific. This contrast between closely related lineages reveals one population diverging due primarily to geographic isolation and genetic drift, and the other achieving taxonomic species status under the influence of selection.
Disentangling the complex evolutionary history of the Western Palearctic blue tits (Cyanistes spp.) – phylogenomic analyses suggest radiation by multiple colonization events and subsequent isolation
Authors: Stervander Martin , Illera Juan Carlos , Kvist Laura , Barbosa Pedro , Keehnen Naomi P. , Pruisscher Peter , Bensch Staffan , Hansson Bengt
Published: March 2015

Isolated islands and their often unique biota continue to play key roles for understanding the importance of drift, genetic variation and adaptation in the process of population differentiation and speciation. One island system that has inspired and intrigued evolutionary biologists is the blue tit complex (Cyanistes spp.) in Europe and Africa, in particular the complex evolutionary history of the multiple genetically distinct taxa of the Canary Islands. Understanding Afrocanarian colonization events is of particular importance because of recent unconventional suggestions that these island populations acted as source of the widespread population in mainland Africa. We investigated the relationship between mainland and island blue tits using a combination of Sanger sequencing at a population level (20 loci; 12 500 nucleotides) and next‐generation sequencing of single population representatives (>3 200 000 nucleotides), analysed in coalescence and phylogenetic frameworks. We found (i) that Afrocanarian blue tits are monophyletic and represent four major clades, (ii) that the blue tit complex has a continental origin and that the Canary Islands were colonized three times, (iii) that all island populations have low genetic variation, indicating low long‐term effective population sizes and (iv) that populations on La Palma and in Libya represent relicts of an ancestral North African population. Further, demographic reconstructions revealed (v) that the Canary Islands, conforming to traditional views, hold sink populations, which have not served as source for back colonization of the African mainland. Our study demonstrates the importance of complete taxon sampling and an extensive multimarker study design to obtain robust phylogeographical inferences.
Heterogeneity of genetic architecture of body size traits in a free‐living population
Authors: Bérénos Camillo , Ellis Philip A. , Pilkington Jill G. , Lee S. Hong , Gratten Jake , Pemberton Josephine M.
Published: March 2015

Knowledge of the underlying genetic architecture of quantitative traits could aid in understanding how they evolve. In wild populations, it is still largely unknown whether complex traits are polygenic or influenced by few loci with major effect, due to often small sample sizes and low resolution of marker panels. Here, we examine the genetic architecture of five adult body size traits in a free‐living population of Soay sheep on St Kilda using 37 037 polymorphic SNPs. Two traits (jaw and weight) show classical signs of a polygenic trait: the proportion of variance explained by a chromosome was proportional to its length, multiple chromosomes and genomic regions explained significant amounts of phenotypic variance, but no SNPs were associated with trait variance when using GWAS. In comparison, genetic variance for leg length traits (foreleg, hindleg and metacarpal) was disproportionately explained by two SNPs on chromosomes 16 (s23172.1) and 19 (s74894.1), which each explained >10% of the additive genetic variance. After controlling for environmental differences, females heterozygous for s74894.1 produced more lambs and recruits during their lifetime than females homozygous for the common allele conferring long legs. We also demonstrate that alleles conferring shorter legs have likely entered the population through a historic admixture event with the Dunface sheep. In summary, we show that different proxies for body size can have very different genetic architecture and that dense SNP helps in understanding both the mode of selection and the evolutionary history at loci underlying quantitative traits in natural populations.
Niche partitioning in arbuscular mycorrhizal communities in temperate grasslands: a lesson from adjacent serpentine and nonserpentine habitats
Authors: Kohout Petr , Doubková Pavla , Bahram Mohammad , Suda Jan , Tedersoo Leho , Voříšková Jana , Sudová Radka
Published: March 2015

Arbuscular mycorrhizal fungi (AMF) represent an important soil microbial group playing a fundamental role in many terrestrial ecosystems. We explored the effects of deterministic (soil characteristics, host plant life stage, neighbouring plant communities) and stochastic processes on AMF colonization, richness and community composition in roots of Knautia arvensis (Dipsacaceae) plants from three serpentine grasslands and adjacent nonserpentine sites. Methodically, the study was based on 454‐sequencing of the ITS region of rDNA. In total, we detected 81 molecular taxonomical operational units (MOTUs) belonging to the Glomeromycota. Serpentine character of the site negatively influenced AMF root colonization, similarly as higher Fe concentration. AMF MOTUs richness linearly increased along a pH gradient from 3.5 to 5.8. Contrary, K and Cr soil concentration had a negative influence on AMF MOTUs richness. We also detected a strong relation between neighbouring plant community composition and AMF MOTUs richness. Although spatial distance between the sampled sites (c. 0.3–3 km) contributed to structuring AMF communities in K. arvensis roots, environmental parameters were key factors in this respect. In particular, the composition of AMF communities was shaped by the complex of serpentine conditions, pH and available soil Ni concentration. The composition of AMF communities was also dependent on host plant life stage (vegetative vs. generative). Our study supports the dominance of deterministic factors in structuring AMF communities in heterogeneous environment composed of an edaphic mosaic of serpentine and nonserpentine soils.
Conserved transcriptional responses to cyanobacterial stressors are mediated by alternate regulation of paralogous genes in Daphnia
Authors: Asselman Jana , Pfrender Michael E. , Lopez Jacqueline A. , De Coninck Dieter I. M. , Janssen Colin R. , Shaw Joseph R. , De Schamphelaere Karel A. C.
Published: March 2015

Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles, whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of nonlineage‐specific genes were conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment‐specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families.
High Final Energy of Low‐Level Gallium Arsenide Laser Therapy Enhances Skeletal Muscle Recovery without a Positive Effect on Collagen Remodeling
Authors: de Freitas Carlos Eduardo Assumpção , Bertaglia Raquel Santilone , Vechetti Júnior Ivan José , Mareco Edson Assunção , Salomão Rondinelle Artur Simões , de Paula Tassiana Gutierrez , Nai Gisele Alborghetti , Carvalho Robson Francisco , Pacagnelli Francis Lopes , Dal‐Pai‐Silva Maeli
Published: March 2015

The aim of this study was to evaluate the effects of a Gallium Arsenide (GaAs) laser, using a high final energy of 4.8 J, during muscle regeneration after cryoinjury. Thirty Wistar rats were divided into three groups: Control (C,n = 10); Injured (I,n = 10) and Injured and laser treated (Injured/LLLT,n = 10). The cryoinjury was induced in the central region of the tibialis anterior muscle (TA). The applications of the laser (904 nm, 50 mW average power) were initiated 24 h after injury, at energy density of 69 J cm−1for 48 s, for 5 days, to two points of the lesion. Twenty‐four hours after the final application, theTAmuscle was removed and frozen in liquid nitrogen to assess the general muscle morphology and the gene expression ofTNF‐α,TGF‐β, MyoD, and Myogenin. The Injured/LLLTgroup presented a higher number of regenerating fibers and fewer degenerating fibers (P < 0.05) without changes in the collagen remodeling. In addition, the Injured/LLLTgroup presented a significant decrease in the expression ofTNF‐αand myogenin compared to the injured group (P < 0.05). The results suggest that the GaAs laser, using a high final energy after cryoinjury, promotes muscle recovery without changing the collagen remodeling in the muscle extracellular matrix.
P53‐Mediated Repression of the Reprogramming in Cloned Bovine Embryos Through Direct Interaction with HDAC1 and Indirect Interaction with DNMT3A
Authors: Ma PJ , Zhang H , Li R , Wang YS , Zhang Y , Hua S
Published: March 2015

Contents

P53 is a transcriptional activator, regulating growth arrest, DNA repair and apoptosis. We found that the expression level of P53 and the epigenetic profiles were significantly different in bovine somatic cell nuclear transfer embryos from those in vitro fertilization (IVF) embryos. So we inferred that abnormally expression of P53 might contribute to the incomplete reprogramming. Using bovine foetal fibroblasts, we constructed and screened a highly efficient shRNA vector targeting bovine P53 gene and then reconstituted somatic cell nuclear transfer embryos (RNAi‐SCNT). The results indicated that expression levels of P53 were downregulated significantly in RNAi‐SCNT embryos, and the blastulation rate and the total number of cell increased significantly. Moreover, methylation levels of CpG islands located 5′ region of OCT4, NANOG, H19 and IGF2R in RNAi ‐SCNT embryos were significantly normalized to that IVF embryos, and the methylation levels of genome DNA, H3K9 and H4K5 acetylation levels were also returned to levels similar to the IVF embryos. Differentially expressed genes were identified by microarray, and 28 transcripts were found to be significantly different (> twofolds) in RNAi‐SCNT embryos compared to the control nuclear transfer embryos (SCNT). Among the 28 differentially expressed transcripts, just HDAC1 and DNMT3A were closely associated with the epigenetic modifications. Finally, ChIP further showed that P53 might repress the epigenetic reprogramming by regulating HDAC1 directly and DNMT3A indirectly. These findings offer significant references to further elucidate the mechanism of epigenetic reprogramming in SCNT embryos.
Dengue epidemiology in selected endemic countries: factors influencing expansion factors as estimates of underreporting
Authors: Toan Nguyen T. , Rossi Stefania , Prisco Gabriella , Nante Nicola , Viviani Simonetta
Published: March 2015

Objective

Dengue fever is globally considered underestimated. This study provides expansion factors (EFs) for dengue endemic selected countries and highlights critical issues in the use of EFs.

Methods

We identified dengue epidemiological cohort studies from 2000 to July 2013 through a literature search using PubMed, Web of Science and Lilacs (Latin American and Caribbean Health Sciences Database), pre‐defined keywords and inclusion/exclusion criteria, and included Brazil, Colombia, Nicaragua, Peru, Puerto Rico, Venezuela, Bangladesh, Cambodia, India, Indonesia, Philippines, Singapore, Sri Lanka, Thailand and Vietnam. Dengue national and local passive surveillance data were derived from WHO regional websites, PAHO, SEARO and WPRO. EFs were calculated as CI cohort studies/CI passive data for both national and local levels.

Results

Cohort studies differed in case definition, laboratory test used and surveillance methods. The information on SEARO, PAHO and WPRO websites differed in terms of dengue epidemiological variables, population denominators and completeness. The highest incidence was reported by PAHO countries followed by WPRO and SEARO countries. EFs may vary for the different variables and denominators used for calculation. EFs were the highest in SEARO countries and lowest in PAHO countries. A trend for lower local EFs was observed.

Conclusions

The use of EFs for quantifying dengue underreporting may be problematic due to lack of uniformity in reporting dengue both active and passive surveillance data. Quality dengue surveillance data are urgently needed for a better estimate of dengue disease burden and to measure the impact of preventive intervention.
Psychological interventions for Common Mental Disorders for People Living With HIV in Low‐ and Middle‐Income Countries: systematic review
Authors: Chibanda Dixon , Cowan Frances M. , Healy Jessica L. , Abas Melanie , Lund Crick
Published: March 2015

Objective

To assess the effectiveness of structured psychological interventions against common mental disorders (CMD) in people living with HIV infection (PLWH), in low‐ and middle‐income countries (LMIC).

Methods

Systematic review of psychological interventions for CMD from LMIC for PLWH, with two‐stage screening carried out independently by 2 authors.

Results

Of 190 studies, 5 met inclusion criteria. These were randomised‐controlled trials based on the principles of cognitive behaviour therapy (CBT) and were effective in reducing CMD symptoms in PLWH. Follow‐up of study participants ranged from 6 weeks to 12 months with multiple tools utilised to measure the primary outcome. Four studies showed a high risk of bias, while 1 study from Iran met low risk of bias in all 6 domains of the Cochrane risk of bias tool and all 22 items of the CONSORT instrument.

Conclusion

There is a need for more robust and adequately powered studies to further explore CBT‐based interventions in PLWH. Future studies should report on components of the psychological interventions, fidelity measurement and training, including supervision of delivering agents, particularly where lay health workers are the delivering agent.
Feasibility and effectiveness of two community‐based HIV testing models in rural Swaziland
Authors: Parker Lucy Anne , Jobanputra Kiran , Rusike Lorraine , Mazibuko Sikhathele , Okello Velephi , Kerschberger Bernhard , Jouquet Guillaume , Cyr Joanne , Teck Roger
Published: March 2015

Objectives

To evaluate the feasibility (population reached, costs) and effectiveness (positivity rates, linkage to care) of two strategies of community‐based HIV testing and counselling (HTC) in rural Swaziland.

Methods

Strategies used were mobile HTC (MHTC) and home‐based HTC (HBHTC). Information on age, sex, previous testing and HIV results was obtained from routine HTC records. A consecutive series of individuals testing HIV‐positive were followed up for 6 months from the test date to assess linkage to care.

Results

A total of 9 060 people were tested: 2 034 through MHTC and 7 026 through HBHTC. A higher proportion of children and adolescents (<20 years) were tested through HBHTC than MHTC (57% vs. 17%; P < 0.001). MHTC reached a higher proportion of adult men than HBHTC (42% vs. 39%; P = 0.015). Of 398 HIV‐positive individuals, only 135 (34%) were enrolled in HIV care within 6 months. Of 42 individuals eligible for antiretroviral therapy, 22 (52%) started treatment within 6 months. Linkage to care was lowest among people who had tested previously and those aged 20–40 years. HBHTC was 50% cheaper (US$11 per person tested; $797 per individual enrolled in HIV care) than MHTC ($24 and $1698, respectively).

Conclusion

In this high HIV prevalence setting, a community‐based testing programme achieved high uptake of testing and appears to be an effective and affordable way to encourage large numbers of people to learn their HIV status (particularly underserved populations such as men and young people). However, for community HTC to impact mortality and incidence, strategies need to be implemented to ensure people testing HIV‐positive in the community are linked to HIV care.
‘I was thinking too much’: experiences ofHIV‐positive adults with common mental disorders and poor adherence to antiretroviral therapy in Zimbabwe
Authors: Kidia Khameer , Machando Debra , Bere Tarisai , Macpherson Kirsty , Nyamayaro Primrose , Potter Lucy , Makadzange Tariro , Munjoma Ronald , Marufu Marshall , Araya Ricardo , Safren Steven , O'Cleirigh Conall , Chibanda Dixon , Abas Melanie
Published: March 2015

Objective

To document the lived experiences of people with both poor mental health and suboptimal adherence to antiretroviral therapy in highHIVprevalence settings.

Methods

In‐depth qualitative interviews were conducted with 47 (female = 31)HIV‐positive adults who scored above the cut‐point on a locally validated scale for common mental disorders (CMDs). Purposive sampling was used to recruit participants with evidence of poor adherence. Six additional key informant interviews (female = 6) were conducted with healthcare workers. Data were collected and analysed inductively by an interdisciplinary coding team.

Results

The major challenges faced by participants were stressors (poverty, stigma, marital problems) and symptoms of CMDs (‘thinking too much’, changes to appetite and sleep, ‘burdened heart’ and low energy levels). Thinking too much, which appears closely related to rumination, was the symptom with the greatest negative impact on adherence to antiretroviral therapy amongHIV‐positive adults with CMDs. In turn, thinking too much was commonly triggered by the stressors faced by people living withHIV/AIDS, especially poverty. Finally, participants desired private counselling, access to income‐generating activities and family engagement in mental health care.

Conclusions

Better understanding of the local expression of mental disorders and of underlying stressors can inform the development of culturally sensitive interventions to reduce CMDs and poor adherence to antiretroviral therapy.
OsNAC2 encoding a NAC transcription factor that affects plant height through mediating the gibberellic acid pathway in rice
Authors: Chen Xu , Lu Songchong , Wang Yaofeng , Zhang Xuan , Lv Bo , Luo Liqiong , Xi Dandan , Shen Jiabin , Ma Hong , Ming Feng
Published: March 2015

Plant height and flowering time are key agronomic traits affecting yield in rice (Oryza sativa). In this study, we investigated the functions in rice growth and development of OsNAC2, encoding a NAC transcription factor in rice. Transgenic plants that constitutively expressed OsNAC2 had shorter internodes, shorter spikelets, and were more insensitive to gibberellic acid (GA3). In addition, the levels of GAs decreased in OsNAC2 overexpression plants, compared with the wild‐type. Moreover, flowering was delayed for approximately 5 days in transgenic lines. The transcription of Hd3a, a flowering‐time related gene, was suppressed in transgenic lines. In addition, transgenic Arabidopsis plants expressing OsNAC2 were also more insensitive to GA3. The expression levels of GA biosynthetic genes OsKO2 and OsKAO were repressed. The expression of OsSLRL, encoding a repressor in the GA signal pathway, and OsEATB, which encodes a repressor of GA biosynthesis, were both enhanced. Western blotting indicated that DELLA also accumulated at the protein level. Dual‐luciferase reporter analyses, yeast one‐hybrid assays and ChIP‐qPCR suggested that OsNAC2 directly interacted with the promoter of OsEATB and OsKO2. Taken together, we proposed that OsNAC2 is a negative regulator of the plant height and flowering time, which acts by directly regulating key genes of the GA pathway in rice.
Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther, a MYB domain protein
Authors: Zhu Xiaolei , Liang Wanqi , Cui Xiao , Chen Mingjiao , Yin Changsong , Luo Zhijing , Zhu Jiaying , Lucas William J. , Wang Zhiyong , Zhang Dabing
Published: March 2015

Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over‐expression of the BR‐synthesis gene D11 or a BR‐signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non‐transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop.
Effects of VEGF and MSCs on vascular regeneration in a trauma model in rats
Authors: Niyaz Mehmet , Gürpınar Özer Aylin , Oktar Gürsel Levent , Günaydın Serdar , Onur Mehmet Ali , Özsin Kadir Kaan , Yener Ali
Published: March 2015

In the human body, vascular injuries that are caused by trauma, vessel lumen stenosis, and occlusions are often irreversible and can lead to sequelae formation as the vessels cannot reproduce fast enough. To solve this problem, the blood flow must be returned to the region as fast as possible. The adipose tissue contains progenitor cells with angiogenic potential and can be used to resolve the issue.

In the present study, mesenchymal stem cells (MSCs) derived from rat adipose tissue, vascular endothelial growth factor (VEGF), and their mixture were applied on the dorsum of a rat, which was traumatized and its contribution to vascular regeneration was reviewed. No application was made to the control group.

The results showed that the percentage of necrotic area was significantly lower in the MSC group than that of all the other groups. When the VEGF group was compared to the VEGF + MSCs, the percentage of necrotic area was observed to be similiar. However, VEGF showed effects only when a large quantites of VEGF was applied to the flap area.

VEGF could not fully respond to the needs, whereas MSCs can produce VEGF according to the needs of tissue. This makes them superior to stem cells.
Observation of the simplest Criegee intermediate CH
2
OO in the gas-phase ozonolysis of ethylene
Authors: Womack Caroline C. , Martin-Drumel Marie-Aline , Brown Gordon G. , Field Robert W. , McCarthy Michael C.
Published: March 2015

Trace amounts of the simplest Criegee intermediate in the gas-phase ozonolysis of ethylene are detected by high-resolution spectroscopy.
Pb pollution from leaded gasoline in South America in the context of a 2000-year metallurgical history
Authors: Eichler Anja , Gramlich Gabriela , Kellerhals Thomas , Tobler Leonhard , Schwikowski Margit
Published: March 2015

Ice core records show that anthropogenic Pb pollution levels from road traffic in South America exceed those of any historical metallurgy.
Near-infrared–driven decomposition of metal precursors yields amorphous electrocatalytic films
Authors: Salvatore Danielle A. , Dettelbach Kevan E. , Hudkins Jesse R. , Berlinguette Curtis P.
Published: March 2015

A method for creating electrocatalyst films extends the scope of usable substrates to non-conducting and three-dimensional electrodes.
The search for signs of life on exoplanets at the interface of chemistry and planetary science
Authors: Seager Sara , Bains William
Published: March 2015

Present theory and future opportunities in the search for biosignature gases in exoplanet atmospheres are reviewed.
Lactococcal 949 Group Phages Recognize a Carbohydrate Receptor on the Host Cell Surface
Authors: Mahony Jennifer , Randazzo Walter , Neve Horst , Settanni Luca , van Sinderen Douwe
Published: March 2015

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages.
Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides
Authors: Oehlenschlæger Christian Berg , Løvgreen Monika Nøhr , Reinauer Eva , Lehtinen Emilia , Pind Marie-Louise Lindberg , Harris Pernille , Martinussen Jan , Willemoës Martin
Published: March 2015

Analysis of the genome of
Bacillus halodurans
strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the
comEB
gene and the
dcdB
gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in
B. halodurans
, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both
comEB
and
dcdB
were cloned, overexpressed in
Escherichia coli
, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the
B. halodurans
enzyme resembled the
Mycobacterium tuberculosis
enzyme the most. An investigation of sequenced genomes from other species of the genus
Bacillus
revealed that not only the genome of
B. halodurans
but also the genomes of
Bacillus pseudofirmus
,
Bacillus thuringiensis
,
Bacillus hemicellulosilyticus
,
Bacillus marmarensis
,
Bacillus cereus
, and
Bacillus megaterium
encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight
dcdB
homologs from
Bacillus
species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.
Metabolic Profiling of Geobacter sulfurreducens during Industrial Bioprocess Scale-Up
Authors: Muhamadali Howbeer , Xu Yun , Ellis David I. , Allwood J. William , Rattray Nicholas J. W. , Correa Elon , Alrabiah Haitham , Lloyd Jonathan R. , Goodacre Royston
Published: March 2015

During the industrial scale-up of bioprocesses it is important to establish that the biological system has not changed significantly when moving from small laboratory-scale shake flasks or culturing bottles to an industrially relevant production level. Therefore, during upscaling of biomass production for a range of metal transformations, including the production of biogenic magnetite nanoparticles by
Geobacter sulfurreducens
, from 100-ml bench-scale to 5-liter fermentors, we applied Fourier transform infrared (FTIR) spectroscopy as a metabolic fingerprinting approach followed by the analysis of bacterial cell extracts by gas chromatography-mass spectrometry (GC-MS) for metabolic profiling. FTIR results clearly differentiated between the phenotypic changes associated with different growth phases as well as the two culturing conditions. Furthermore, the clustering patterns displayed by multivariate analysis were in agreement with the turbidimetric measurements, which displayed an extended lag phase for cells grown in a 5-liter bioreactor (24 h) compared to those grown in 100-ml serum bottles (6 h). GC-MS analysis of the cell extracts demonstrated an overall accumulation of fumarate during the lag phase under both culturing conditions, coinciding with the detected concentrations of oxaloacetate, pyruvate, nicotinamide, and glycerol-3-phosphate being at their lowest levels compared to other growth phases. These metabolites were overlaid onto a metabolic network of
G. sulfurreducens
, and taking into account the levels of these metabolites throughout the fermentation process, the limited availability of oxaloacetate and nicotinamide would seem to be the main metabolic bottleneck resulting from this scale-up process. Additional metabolite-feeding experiments were carried out to validate the above hypothesis. Nicotinamide supplementation (1 mM) did not display any significant effects on the lag phase of
G. sulfurreducens
cells grown in the 100-ml serum bottles. However, it significantly improved the growth behavior of cells grown in the 5-liter bioreactor by reducing the lag phase from 24 h to 6 h, while providing higher yield than in the 100-ml serum bottles.
A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters
Authors: Esteban-Torres María , Landete José María , Reverón Inés , Santamaría Laura , de las Rivas Blanca , Muñoz Rosario
Published: March 2015

Lactobacillus plantarum
is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant.
L. plantarum
strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28
L. plantarum
strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the
est_1092
gene. When the
est_1092
gene was introduced into
L. plantarum
WCFS1 or
L. lactis
MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the
L. plantarum
strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of
est_1092
was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some
L. plantarum
strains provides them with additional advantages to survive and grow on plant environments.
Two Phages, phiIPLA-RODI and phiIPLA-C1C, Lyse Mono- and Dual-Species Staphylococcal Biofilms
Authors: Gutiérrez Diana , Vandenheuvel Dieter , Martínez Beatriz , Rodríguez Ana , Lavigne Rob , García Pilar
Published: March 2015

Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the
Myoviridae
family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of
Spounavirinae
members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the
Twort-like virus
genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of
Staphylococcus aureus
and
S. epidermidis
with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 × 10
−7
± 2.34 × 10
−9
and 1.1 × 10
−7
± 2.08 × 10
−9
, respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.
Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production
Authors: Maleki Susan , Mærk Mali , Valla Svein , Ertesvåg Helga
Published: March 2015

The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium
Pseudomonas fluorescens
relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in
P. fluorescens
. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of
zwf-1
resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of
gcd
increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.
New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli
Authors: Schwendener Sybille , Perreten Vincent
Published: March 2015

Four
Staphylococcus aureus
-
Escherichia coli
shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-P

cap

-HC and pTSSCm-P

cap

contain the strong constitutive promoter of
S. aureus
type 1 capsule gene 1A (P

cap

) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (
rgs
-
his
6
). All plasmids contained the backbone derived from pBUS1, including the
E. coli
origin ColE1, five copies of terminator
rrnB
T
1
, and tetracycline resistance marker
tet
(L) for
S. aureus
and
E. coli
. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin
oriL
from pUB110 (pBUS1-HC and pBUS1-P

cap

-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-P

cap

). The new constructs displayed increased plasmid yield and segregational stability in
S. aureus
. Furthermore, pBUS1-P

cap

-HC and pTSSCm-P

cap

offer the potential to generate C-terminal RGS-His
6
translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the
rgs
-
his
6
codons to the 3′ end of the target gene. The generation of the
rgs
-
his
6
codon-fusion, gene expression, and protein purification were demonstrated in both
S. aureus
and
E. coli
using the macrolide-lincosamide-streptogramin B resistance gene
erm
(44) inserted downstream of P

cap

. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.
Novel
bla
ROB-1
-Bearing Plasmid Conferring Resistance to β-Lactams in Haemophilus parasuis Isolates from Healthy Weaning Pigs
Authors: Moleres Javier , Santos-López Alfonso , Lázaro Isidro , Labairu Javier , Prat Cristina , Ardanuy Carmen , González-Zorn Bruno , Aragon Virginia , Garmendia Junkal
Published: March 2015

Haemophilus parasuis
, the causative agent of Glässer's disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in
H. parasuis
. Prediction of
H. parasuis
virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with
H. parasuis
virulence, such as
lsgB
and group 1
vtaA
, while biofilm growth has been associated with nonvirulent strains. In this study, 86
H. parasuis
nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of
lsgB
and group 1
vtaA
, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the
bla
ROB-1
β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the
Pasteurellaceae
, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine
H. parasuis
nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer's disease or in nontypeable
Haemophilus influenzae
isolates. Altogether, we show that commensal
H. parasuis
isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.
Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase
Authors: Price Claire L. , Warrilow Andrew G. S. , Parker Josie E. , Mullins Jonathan G. L. , Nes W. David , Kelly Diane E. , Kelly Steven L.
Published: March 2015

Mycosphaerella graminicola
(
Zymoseptoria tritici
) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The
M. graminicola
CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with
Candida albicans
CYP51 (CaCYP51) and that of eburicol only with
M. graminicola
CYP51 (MgCYP51). The availability of the functional
in vivo
redox partner enabled the
in vitro
catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC
50
) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.
Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii
Authors: Oh Man Hwan , Lee Je Chul , Kim Jungmin , Choi Chul Hee , Han Kyudong
Published: March 2015

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant
Acinetobacter baumannii
. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of
A. baumannii
merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in
A. baumannii
, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by
A. baumannii
ATCC 19606 and to
ompA
gene deletion in other
A. baumannii
strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant
A. baumannii
.
Integrated Kinetic and Probabilistic Modeling of the Growth Potential of Bacterial Populations
Authors: George S. M. , Métris A. , Baranyi J.
Published: March 2015

When bacteria are exposed to osmotic stress, some cells recover and grow, while others die or are unculturable. This leads to a viable count growth curve where the cell number decreases before the onset of the exponential growth phase. From such curves, it is impossible to estimate what proportion of the initial cells generates the growth because it leads to an ill-conditioned numerical problem. Here, we applied a combination of experimental and statistical methods, based on optical density measurements, to infer both the probability of growth and the maximum specific growth rate of the culture. We quantified the growth potential of a bacterial population as a quantity composed from the probability of growth and the “suitability” of the growing subpopulation to the new environment. We found that, for all three laboratory media studied, the probability of growth decreased while the “work to be done” by the growing subpopulation (defined as the negative logarithm of their suitability parameter) increased with NaCl concentration. The results suggest that the effect of medium on the probability of growth could be described by a simple shift parameter, a differential NaCl concentration that can be accounted for by the change in the medium composition. Finally, we highlighted the need for further understanding of the effect of the osmoprotectant glycine betaine on metabolism.
Accumulation of
d
-Glucose from Pentoses by Metabolically Engineered Escherichia coli
Authors: Xia Tian , Han Qi , Costanzo William V. , Zhu Yixuan , Urbauer Jeffrey L. , Eiteman Mark A.
Published: March 2015

Escherichia coli
that is unable to metabolize
d
-glucose (with knockouts in
ptsG
,
manZ
, and
glk
) accumulates a small amount of
d
-glucose (yield of about 0.01 g/g) during growth on the pentoses
d
-xylose or
l
-arabinose as a sole carbon source. Additional knockouts in the
zwf
and
pfkA
genes, encoding, respectively,
d
-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (
E. coli
MEC143), increased accumulation to greater than 1 g/liter
d
-glucose and 100 mg/liter
d
-mannose from 5 g/liter
d
-xylose or
l
-arabinose. Knockouts of other genes associated with interconversions of
d
-glucose-phosphates demonstrate that
d
-glucose is formed primarily by the dephosphorylation of
d
-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter
d
-xylose, MEC143 generated 4.4 g/liter
d
-glucose and 0.6 g/liter
d
-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.