AEM Current Issue Applied and Environmental Microbiology RSS feed 1098-5336 June 2019 Applied and Environmental Microbiology 0099-2240 <![CDATA[High Prevalence of Fluoroquinolone-Resistant Campylobacter Bacteria in Sheep and Increased Campylobacter Counts in the Bile and Gallbladders of Sheep Medicated with Tetracycline in Feed [Food Microbiology]]]> Campylobacter is a major foodborne pathogen in humans and a significant cause of abortion in sheep. Although ruminants are increasingly recognized as important reservoirs for Campylobacter species, limited information is available about the molecular epidemiology and antimicrobial resistance (AMR) profiles of sheep Campylobacter. Here, we describe a two-trial study that examined Campylobacter profiles in sheep and determined whether in-feed tetracycline (TET) influenced the distribution and AMR profiles of Campylobacter. Each trial involved 80 commercial sheep naturally infected with Campylobacter: 40 of these sheep were medicated with tetracycline in feed, while the other 40 received feed without antibiotics. Fecal and bile samples were collected for the isolation of Campylobacter. The bacterial isolates were analyzed for antimicrobial susceptibility and genotypes. The results revealed that 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter (Campylobacter jejuni and Campylobacter coli), with no significant differences between the medicated and nonmedicated groups. All but one of the tested Campylobacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resistance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant to FQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coli ST902 were the predominant genotypes in the sheep. Feed medication with tetracycline did not affect the overall prevalence, species distribution, and AMR profiles of Campylobacter, but it did increase the total Campylobacter counts in bile and gallbladder. These findings identify predominant Campylobacter clones, reveal the high prevalence of FQ-resistant C. coli, and provide new insights into the epidemiology of Campylobacter in sheep.

IMPORTANCE Campylobacter is a major cause of foodborne illness in humans, and antibiotic-resistant Campylobacter is considered a serious threat to public health in the United States and worldwide. As a foodborne pathogen, Campylobacter commonly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Results from this study reveal the predominant genotypes and high prevalence of tetracycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter. The finding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as this class of antibiotics is not used for sheep in the United States, and it may suggest the transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep. Additionally, the results demonstrate that in-feed medication with tetracycline increases Campylobacter counts in gallbladders, suggesting that the antibiotic promotes Campylobacter colonization of the gallbladder. These findings provide new information on Campylobacter epidemiology in sheep, which may be useful for curbing the spread of antibiotic-resistant Campylobacter in animal reservoirs.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00008-19 hwp:master-id:aem;AEM.00008-19 American Society for Microbiology 2019-06-01 Food Microbiology 85 11 e00008-19 e00008-19 hw_mjid:aem;85/11/e00008-19
<![CDATA[Phototrophic Lactate Utilization by Rhodopseudomonas palustris Is Stimulated by Coutilization with Additional Substrates [Physiology]]]> The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris is known for its metabolic versatility and is of interest for various industrial and environmental applications. Despite decades of research on R. palustris growth under diverse conditions, patterns of R. palustris growth and carbon utilization with mixtures of carbon substrates remain largely unknown. R. palustris readily utilizes most short-chain organic acids but cannot readily use lactate as a sole carbon source. Here we investigated the influence of mixed-substrate utilization on phototrophic lactate consumption by R. palustris. We found that lactate was simultaneously utilized with a variety of other organic acids and glycerol in time frames that were insufficient for R. palustris growth on lactate alone. Thus, lactate utilization by R. palustris was expedited by its coutilization with additional substrates. Separately, experiments using carbon pairs that did not contain lactate revealed acetate-mediated inhibition of glycerol utilization in R. palustris. This inhibition was specific to the acetate-glycerol pair, as R. palustris simultaneously utilized acetate or glycerol when either was paired with succinate or lactate. Overall, our results demonstrate that (i) R. palustris commonly employs simultaneous mixed-substrate utilization, (ii) mixed-substrate utilization expands the spectrum of readily utilized organic acids in this species, and (iii) R. palustris has the capacity to exert carbon catabolite control in a substrate-specific manner.

IMPORTANCE Bacterial carbon source utilization is frequently assessed using cultures provided single carbon sources. However, the utilization of carbon mixtures by bacteria (i.e., mixed-substrate utilization) is of both fundamental and practical importance; it is central to bacterial physiology and ecology, and it influences the utility of bacteria as biotechnology. Here we investigated mixed-substrate utilization by the model organism Rhodopseudomonas palustris. Using mixtures of organic acids and glycerol, we show that R. palustris exhibits an expanded range of usable carbon substrates when provided substrates in mixtures. Specifically, coutilization enabled the prompt consumption of lactate, a substrate that is otherwise not readily used by R. palustris. Additionally, we found that R. palustris utilizes acetate and glycerol sequentially, revealing that this species has the capacity to use some substrates in a preferential order. These results provide insights into R. palustris physiology that will aid the use of R. palustris for industrial and commercial applications.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00048-19 hwp:master-id:aem;AEM.00048-19 American Society for Microbiology 2019-06-01 Physiology 85 11 e00048-19 e00048-19 hw_mjid:aem;85/11/e00048-19
<![CDATA[Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii [Genetics and Molecular Biology]]]> The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii. In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii. In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application.

IMPORTANCE Acinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00066-19 hwp:master-id:aem;AEM.00066-19 American Society for Microbiology 2019-06-01 Genetics and Molecular Biology 85 11 e00066-19 e00066-19 hw_mjid:aem;85/11/e00066-19
<![CDATA[Editorial Board [Masthead]]]> 2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.masthead.85-11 hwp:resource-id:aem;85/11/masthead-85-11 American Society for Microbiology 2019-06-01 Masthead 85 11 masthead-85-11 masthead-85-11 hw_mjid:aem;85/11/masthead-85-11 <![CDATA[Influence of Plant Species, Tissue Type, and Temperature on the Capacity of Shiga-Toxigenic Escherichia coli To Colonize, Grow, and Be Internalized by Plants [Food Microbiology]]]> Contamination of fresh produce with pathogenic Escherichia coli, including Shiga-toxigenic E. coli (STEC), represents a serious risk to human health. Colonization is governed by multiple bacterial and plant factors that can impact the probability and suitability of bacterial growth. Thus, we aimed to determine whether the growth potential of STEC for plants associated with foodborne outbreaks (two leafy vegetables and two sprouted seed species) is predictive of the colonization of living plants, as assessed from growth kinetics and biofilm formation in plant extracts. The fitness of STEC isolates was compared to that of environmental E. coli isolates at temperatures relevant to plant growth. Growth kinetics in plant extracts varied in a plant-dependent and isolate-dependent manner for all isolates, with spinach leaf lysates supporting the highest rates of growth. Spinach extracts also supported the highest levels of biofilm formation. Saccharides were identified to be the major driver of bacterial growth, although no single metabolite could be correlated with growth kinetics. The highest level of in planta colonization occurred on alfalfa sprouts, though internalization was 10 times more prevalent in the leafy vegetables than in sprouted seeds. Marked differences in in planta growth meant that the growth potential of STEC could be inferred only for sprouted seeds. In contrast, biofilm formation in extracts related to spinach colonization. Overall, the capacity of E. coli to colonize, grow, and be internalized within plants or plant-derived matrices was influenced by the isolate type, plant species, plant tissue type, and temperature, complicating any straightforward relationship between in vitro and in planta behaviors.

IMPORTANCE Fresh produce is an important vehicle for STEC transmission, and experimental evidence shows that STEC can colonize plants as secondary hosts, but differences in the capacity to colonize occur between different plant species and tissues. Therefore, an understanding of the impact that these plant factors have on the ability of STEC to grow and establish is required for food safety considerations and risk assessment. Here, we determined whether growth and the ability of STEC to form biofilms in plant extracts could be related to specific plant metabolites or could predict the ability of the bacteria to colonize living plants. Growth rates for sprouted seeds (alfalfa and fenugreek) but not those for leafy vegetables (lettuce and spinach) exhibited a positive relationship between plant extracts and living plants. Therefore, the detailed variations at the level of the bacterial isolate, plant species, and tissue type all need to be considered in risk assessment.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00123-19 hwp:master-id:aem;AEM.00123-19 American Society for Microbiology 2019-06-01 Food Microbiology 85 11 e00123-19 e00123-19 hw_mjid:aem;85/11/e00123-19
<![CDATA[Rapid and Stable Microbial Community Assembly in the Headwaters of a Third-Order Stream [Microbial Ecology]]]> Small streams and their headwaters are key sources of microbial diversity in fluvial systems and serve as an entry point for bacteria from surrounding environments. Community assembly processes occurring in these streams shape downstream population structure and nutrient cycles. To elucidate the development and stability of microbial communities along the length of a first- through third-order stream, fine-scale temporal and spatial sampling regimes were employed along McNutt Creek in Athens, GA, USA. 16S rRNA amplicon libraries were constructed from samples collected on a single day from 19 sites spanning the first 16.76 km of the stream. To provide context for this spatial study and evaluate temporal variability, selected sites at the stream’s upper, mid, and lower reaches were sampled daily for 5 days preceding and following the spatial study. In a second study, three sites at and near the creek’s headwaters were sampled daily for 11 days to understand initial bacterioplankton community assembly. Both studies revealed decreasing alpha and beta diversity with increasing downstream distance. These trends were accompanied by the enrichment of a small fraction of taxa found at low abundance in headwater-proximal sites. Similar sets of taxa consistently increased in relative abundance in downstream samples over time scales ranging from 1 day to 1 year, many of which belong to clades known to be abundant in freshwater environments. These results underpin the importance of headwaters as the site of rapid in-stream selection that results in the reproducible establishment of a highly stable community of freshwater riverine bacteria.

IMPORTANCE Headwater streams are critical introduction points of microbial diversity for larger connecting rivers and play key roles in the establishment of taxa that partake in in-stream nutrient cycling. We examined the microbial community composition of a first- through third-order stream using fine-scale temporal and spatial regimes. Our results show that the bacterioplankton community develops rapidly and predictably from the headwater population with increasing total stream length. Along the length of the stream, the microbial community exhibits substantial diversity loss and enriches repeatedly for select taxa across days and years, although the relative abundances of individual taxa vary over time and space. This repeated enrichment of a stable stream community likely contributes to the stability and flexibility of downstream communities.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00188-19 hwp:master-id:aem;AEM.00188-19 American Society for Microbiology 2019-06-01 Microbial Ecology 85 11 e00188-19 e00188-19 hw_mjid:aem;85/11/e00188-19
<![CDATA[Determinants of Phage Host Range in Staphylococcus Species [Minireviews]]]> Bacteria in the genus Staphylococcus are important targets for phage therapy due to their prevalence as pathogens and increasing antibiotic resistance. Here we review Staphylococcus outer surface features and specific phage resistance mechanisms that define the host range, the set of strains that an individual phage can potentially infect. Phage infection goes through five distinct phases: attachment, uptake, biosynthesis, assembly, and lysis. Adsorption inhibition, encompassing outer surface teichoic acid receptor alteration, elimination, or occlusion, limits successful phage attachment and entry. Restriction-modification systems (in particular, type I and IV systems), which target phage DNA inside the cell, serve as the major barriers to biosynthesis as well as transduction and horizontal gene transfer between clonal complexes and species. Resistance to late stages of infection occurs through mechanisms such as assembly interference, in which staphylococcal pathogenicity islands siphon away superinfecting phage proteins to package their own DNA. While genes responsible for teichoic acid biosynthesis, capsule, and restriction-modification are found in most Staphylococcus strains, a variety of other host range determinants (e.g., clustered regularly interspaced short palindromic repeats, abortive infection, and superinfection immunity) are sporadic. The fitness costs of phage resistance through teichoic acid structure alteration could make staphylococcal phage therapies promising, but host range prediction is complex because of the large number of genes involved, and the roles of many of these are unknown. In addition, little is known about the genetic determinants that contribute to host range expansion in the phages themselves. Future research must identify host range determinants, characterize resistance development during infection and treatment, and examine population-wide genetic background effects on resistance selection.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00209-19 hwp:master-id:aem;AEM.00209-19 American Society for Microbiology 2019-06-01 Minireviews 85 11 e00209-19 e00209-19 hw_mjid:aem;85/11/e00209-19
<![CDATA[Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase [Enzymology and Protein Engineering]]]> Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMORp) with a high affinity toward aliphatic methyl ketones (Km < 3.0 μM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (kcat = 2.1 s–1; Km = 1.5 μM). Furthermore, BVMORp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMORp as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter–1 h–1. Thus, BVMORp is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation.

IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMORp) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMORp possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMORp-catalyzed oxidation holds great promise to replace traditional fermentation.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00239-19 hwp:master-id:aem;AEM.00239-19 American Society for Microbiology 2019-06-01 Enzymology and Protein Engineering 85 11 e00239-19 e00239-19 hw_mjid:aem;85/11/e00239-19
<![CDATA[Microbial Community Analysis Provides Insights into the Effects of Tetrahydrofuran on 1,4-Dioxane Biodegradation [Biodegradation]]]> Tetrahydrofuran (THF) is known to induce the biodegradation of 1,4-dioxane (dioxane), an emerging contaminant, but the mechanisms by which THF affects dioxane biodegradation in microbial communities are not well understood. To fill this knowledge gap, changes in the microbial community structure in microcosm experiments with synthetic medium and landfill leachate were examined over time using 16S rRNA gene amplicon sequencing and functional gene quantitative PCR assays. The overarching hypothesis being tested was that THF promoted dioxane biodegradation by increasing the abundance of dioxane-degrading bacteria in the consortium. The data revealed that in experiments with synthetic medium, the addition of THF significantly increased the abundance of Pseudonocardia, a genus with several representatives that can grow on both dioxane and THF, and of Rhodococcus ruber, a species that can use THF as the primary growth substrate while cometabolizing dioxane. However, in similar experiments with landfill leachate, only R. ruber was significantly enriched. When the THF concentration was higher than the dioxane concentration, THF competitively inhibited dioxane degradation since dioxane degradation was negligible, while the dioxane-degrading bacteria and the corresponding THF/dioxane monooxygenase gene copies increased by a few orders of magnitude.

IMPORTANCE Widespread in groundwater and carcinogenic to humans, 1,4-dioxane (dioxane) is attracting significant attention in recent years. Advanced oxidation processes can effectively remove dioxane but require high energy consumption and operation costs. Biological removal of dioxane is of particular interest due to the ability of some bacteria to mineralize dioxane at a low energy cost. Although dioxane is generally considered recalcitrant to biodegradation, more than 20 types of bacteria can degrade dioxane as the sole electron donor substrate or the secondary electron donor substrate. In the latter case, tetrahydrofuran (THF) is commonly studied as the primary electron donor substrate. Previous work has shown that THF promotes dioxane degradation at a low THF concentration but inhibits dioxane degradation at a high THF concentration. Our work expanded on the previous work by mechanically examining the effects of THF on dioxane degradation in a microbial community context.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00244-19 hwp:master-id:aem;AEM.00244-19 American Society for Microbiology 2019-06-01 Biodegradation 85 11 e00244-19 e00244-19 hw_mjid:aem;85/11/e00244-19
<![CDATA[Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG [Biotechnology]]]> Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.

IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00329-19 hwp:master-id:aem;AEM.00329-19 American Society for Microbiology 2019-06-01 Biotechnology 85 11 e00329-19 e00329-19 hw_mjid:aem;85/11/e00329-19
<![CDATA[Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing [Genetics and Molecular Biology]]]> Methanotrophic bacteria play a crucial role in the Earth’s biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.

IMPORTANCE In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00340-19 hwp:master-id:aem;AEM.00340-19 American Society for Microbiology 2019-06-01 Genetics and Molecular Biology 85 11 e00340-19 e00340-19 hw_mjid:aem;85/11/e00340-19
<![CDATA[Insights into the Phylogeny, Nodule Function, and Biogeographic Distribution of Microsymbionts Nodulating the Orphan Kerstings Groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] in African Soils [Plant Microbiology]]]> Kersting’s groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] is a neglected indigenous African legume adapted to growth in N-deficient soils due to its ability to fix atmospheric N2 via symbiosis with rhizobia. Despite its nutritional and medicinal uses, to date there is little information on the phylogeny and functional traits of its microsymbionts, aspects that are much needed for its conservation and improvement. This study explored the morphogenetic diversity, phylogenetic relationships, and N2-fixing efficiency of Kersting’s groundnut rhizobial isolates from contrasting environments in Ghana, South Africa, and Mozambique. BOX-PCR fingerprinting revealed high diversity among the rhizobial populations, which was influenced by geographic origin. Of the 164 isolates evaluated, 130 BOX-PCR types were identified at a 70% similarity coefficient, indicating that they were not clones. Soil pH and mineral concentrations were found to influence the distribution of bradyrhizobial populations in African soils. Phylogenetic analysis of 16S rRNA genes and multilocus sequence analysis of protein-coding genes (atpD, glnII, gyrB, and rpoB) and symbiotic genes (nifH and nodC) showed that Kersting’s groundnut is primarily nodulated by members of the genus Bradyrhizobium, which are closely related to Bradyrhizobium vignae 7-2T, Bradyrhizobium kavangense 14-3T, Bradyrhizobium subterraneum 58-2-1T, Bradyrhizobium pachyrhizi PAC48T, the type strain of Bradyrhizobium elkanii, and novel groups of Bradyrhizobium species. The bradyrhizobial populations identified exhibited high N2 fixation and induced greater nodulation, leaf chlorophyll concentration, and photosynthetic rates in their homologous host than did the 5 mM KNO3-fed plants and/or the commercial Bradyrhizobium sp. strain CB756, suggesting that they could be good candidates for inoculant formulations upon field testing.

IMPORTANCE Rhizobia play important roles in agroecosystems, where they contribute to improving overall soil health through their symbiotic relationship with legumes. This study explored the microsymbionts nodulating Kersting’s groundnut, a neglected orphan legume. The results revealed the presence of different bradyrhizobial populations with high N2-fixing efficiencies as the dominant symbionts of this legume across diverse agroecologies in Africa. Our findings represent a useful contribution to the literature in terms of the community of microsymbionts nodulating a neglected cultivated legume and its potential for elevation as a major food crop. The presence of potentially novel bradyrhizobial symbionts of Kersting’s groundnut found in this study offers an opportunity for future studies to properly describe, characterize, and delineate these isolates functionally and phylogenetically for use in inoculant production to enhance food/nutritional security.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00342-19 hwp:master-id:aem;AEM.00342-19 American Society for Microbiology 2019-06-01 Plant Microbiology 85 11 e00342-19 e00342-19 hw_mjid:aem;85/11/e00342-19
<![CDATA[Microcin PDI Inhibits Antibiotic-Resistant Strains of Escherichia coli and Shigella through a Mechanism of Membrane Disruption and Protection by Homotrimer Self-Immunity [Environmental Microbiology]]]> Microcin PDI (MccPDI), a class IIa microcin that is produced by Escherichia coli strains 25 and 284, is known to inhibit foodborne pathogenic enterohemorrhagic E. coli serotypes O157:H7 and O26. Here we demonstrate that MccPDI can inhibit Shigella strains and E. coli isolates that are multidrug resistant, the latter including strains known to cause urinary tract infections in people and companion animals. Two exceptions out of 17 strains were identified. One of the two resistant E. coli isolates (AR0349) has a mutation in a critical amino acid residue that was identified in previous work as a requisite for the MccPDI precursor protein (McpM) to interact with outer membrane porin F (OmpF) on susceptible cells. The second resistant E. coli strain (MAD 96) had no mutations in ompF, but it was PCR positive for two antimicrobial peptides, of which colicin Ia/Ib likely inhibits the MccPDI-producing strain during coculture. Recombinant McpM was still effective against strain MAD 96. In an assessment of how MccPDI affects susceptible strains, results from both an extracellular ATP assay and a nucleic acid staining assay were consistent with membrane damage, while the addition of 200- to 600-Da polyethylene glycol (PEG) to cocultures protected against MccPDI (>600-Da PEG did not provide protection). Further studies using a paraformaldehyde cross-linking experiment and a bacterial two-hybrid assay demonstrated that MccPDI immunity protein (McpI) forms a multimeric complex with itself and presumably protects the producer strain from within the periplasm through an unknown mechanism.

IMPORTANCE Microcins represent potential alternatives to conventional antibiotics for human and veterinary medicine. For them to be applied in this manner, however, we need to better understand their spectrum of activity, how these proteins interact with susceptible cells, and how producer cells are protected against the antimicrobial properties of the microcins. For microcin PDI (MccPDI), we report that the spectrum of activity likely includes most E. coli strains due to a conserved binding motif found on an outer membrane protein. Shigella has this motif as well and is susceptible to MccPDI killing via damage to the bacterial membrane. Receptor specificity suggests that these proteins could be used without causing large-scale disruptions to a microbiota, but this also increases the likelihood that resistance can evolve via random mutations. As with conventional antibiotics, good stewardship will be needed to preserve the efficacy of microcins should they be deployed for clinical use.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00371-19 hwp:master-id:aem;AEM.00371-19 American Society for Microbiology 2019-06-01 Environmental Microbiology 85 11 e00371-19 e00371-19 hw_mjid:aem;85/11/e00371-19
<![CDATA[Specific Environmental Temperature and Relative Humidity Conditions and Grafting Affect the Persistence and Dissemination of Salmonella enterica subsp. enterica Serotype Typhimurium in Tomato Plant Tissues [Public and Environmental Health Microbiology]]]> Little is known about the abiotic factors contributing to the preharvest persistence of Salmonella in tomato tissues. Therefore, we investigated the effects of specific environmental conditions and contamination methods on the persistence and dissemination of Salmonella enterica subsp. enterica serotype Typhimurium (JSG626) in tomato plants. When plants were sprayed on the leaves with a JSG626-contaminated solution, JSG626 persistence in the phyllosphere (bacteria located on the surface of the inoculated foliage and stem tissues) was lower at higher temperatures (30°C day/25°C night) than at lower temperatures (20°C day/15°C night). However, wounding cotyledons with contaminated tools improved JSG626 persistence and the internalization rate (2.27%) in planta compared to spray inoculation (0.004%). The systemic dissemination of JSG626 to other tissues increased when contaminated plants were grown under low relative humidity (<40%); however, JSG626 was only detected in the root systems at later sampling times (between 21 and 98 days postinoculation [dpi]). Further, after tomato scions were grafted onto rootstocks using contaminated cutting tools, dissemination of JSG626 was preferentially basipetal and occasionally acropetal in the plants, with higher persistence rates and loads of JSG626 in root systems compared to foliar tissues. JSG626 was detected in the grafting point and root systems up to 242 dpi; however, none of the fruits harvested from contaminated plants between 90 and 137 dpi were positive for JSG626. This study demonstrates that environmental temperature and relative humidity could be good indicators for estimating the persistence of Salmonella enterica in tomato plants. Further, root systems may represent a risk for long-term persistence of Salmonella enterica in tomato plants.

IMPORTANCE Tomatoes are one of the most widely produced vegetables around the world; however, fresh tomatoes have been connected to multiple wide-scale salmonellosis outbreaks over the past decades. Salmonella is commonly found in the environment and can persist in hostile conditions for several weeks before being internalized into plant tissues, where it is protected from conventional sanitation methods. In addition to biotic factors (host, inoculum size, and phytobiome), abiotic factors (environmental conditions) may affect the persistence of Salmonella in crop production. This study demonstrates that specific environmental conditions, the inoculation method, and the inoculum density affect the persistence and dissemination of JSG626 in tomato plant tissues. Our findings enhance the understanding of interactions between Salmonella enterica and fresh produce and may lead to the development of novel management practices on farms.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00403-19 hwp:master-id:aem;AEM.00403-19 American Society for Microbiology 2019-06-01 Public and Environmental Health Microbiology 85 11 e00403-19 e00403-19 hw_mjid:aem;85/11/e00403-19
<![CDATA[Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli [Enzymology and Protein Engineering]]]> Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5'-phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli. The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coli.

IMPORTANCE Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00430-19 hwp:master-id:aem;AEM.00430-19 American Society for Microbiology 2019-06-01 Enzymology and Protein Engineering 85 11 e00430-19 e00430-19 hw_mjid:aem;85/11/e00430-19
<![CDATA[6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33 [Enzymology and Protein Engineering]]]> Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.

IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00454-19 hwp:master-id:aem;AEM.00454-19 American Society for Microbiology 2019-06-01 Enzymology and Protein Engineering 85 11 e00454-19 e00454-19 hw_mjid:aem;85/11/e00454-19
<![CDATA[LanI-Mediated Lantibiotic Immunity in Bacillus subtilis: Functional Analysis [Environmental Microbiology]]]> Lantibiotics subtilin and nisin are produced by Bacillus subtilis and Lactococcus lactis, respectively. To prevent toxicity of their own lantibiotic, both bacteria express specific immunity proteins, called SpaI and NisI. In addition, ABC transporters SpaFEG and NisFEG prevent lantibiotic toxicity by transporting the respective peptides to the extracellular space. Although the three-dimensional structures of SpaI and NisI have been solved, very little is known about the molecular function of either lipoprotein. Using laser-induced liquid bead ion desorption (LILBID)–mass spectrometry, we show here that subtilin interacts with SpaI monomers. The expression of either SpaI or NisI in a subtilin-nonproducing B. subtilis strain resulted in the respective strain being more resistant against either subtilin or nisin. Furthermore, pore formation provided by subtilin and nisin was prevented specifically upon the expression of either SpaI or NisI. As shown with a nisin-subtilin hybrid molecule, the C-terminal part of subtilin but not any particular lanthionine ring was needed for SpaI-mediated immunity. With respect to growth, SpaI provided less immunity against subtilin than is provided by the ABC transporter SpaFEG. However, SpaI prevented pore formation much more efficiently than SpaFEG. Taken together, our data show the physiological function of SpaI as a fast immune response to protect the cellular membrane.

IMPORTANCE The two lantibiotics nisin and subtilin are produced by Lactococcus lactis and Bacillus subtilis, respectively. Both peptides have strong antimicrobial activity against Gram-positive bacteria, and therefore, appropriate protection mechanisms are required for the producing strains. To prevent toxicity of their own lantibiotic, both bacteria express immunity proteins, called SpaI and NisI, and in addition, ABC transporters SpaFEG and NisFEG. Whereas it has been shown that the ABC transporters protect the producing strains by transporting the toxic peptides to the extracellular space, the exact mode of action and the physiological function of the lipoproteins during immunity are still unknown. Understanding the exact role of lantibiotic immunity proteins is of major importance for improving production rates and for the design of newly engineered peptide antibiotics. Here, we show (i) the specificity of each lipoprotein for its own lantibiotic, (ii) the specific physical interaction of subtilin with its lipoprotein SpaI, (iii) the physiological function of SpaI in protecting the cellular membrane, and (iv) the importance of the C-terminal part of subtilin for its interaction with SpaI.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00534-19 hwp:master-id:aem;AEM.00534-19 American Society for Microbiology 2019-06-01 Environmental Microbiology 85 11 e00534-19 e00534-19 hw_mjid:aem;85/11/e00534-19
<![CDATA[Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection [Environmental Microbiology]]]> Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5α. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 µM, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 µM. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs.

IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00694-19 hwp:master-id:aem;AEM.00694-19 American Society for Microbiology 2019-06-01 Environmental Microbiology 85 11 e00694-19 e00694-19 hw_mjid:aem;85/11/e00694-19
<![CDATA[Correction for Gaby et al., "Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline" [Author Correction]]]> 2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00713-19 hwp:resource-id:aem;85/11/e00713-19 American Society for Microbiology 2019-06-01 Author Correction 85 11 e00713-19 e00713-19 hw_mjid:aem;85/11/e00713-19 <![CDATA[Correction for Pitol et al., "Transfer of Enteric Viruses Adenovirus and Coxsackievirus and Bacteriophage MS2 from Liquid to Human Skin" [Author Correction]]]> 2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00721-19 hwp:resource-id:aem;85/11/e00721-19 American Society for Microbiology 2019-06-01 Author Correction 85 11 e00721-19 e00721-19 hw_mjid:aem;85/11/e00721-19 <![CDATA[Articles of Significant Interest in This Issue [Spotlight]]]> 2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.00810-19 hwp:resource-id:aem;85/11/e00810-19 American Society for Microbiology 2019-06-01 Spotlight 85 11 e00810-19 e00810-19 hw_mjid:aem;85/11/e00810-19 <![CDATA[Biofilm-Constructing Variants of Paraburkholderia phytofirmans PsJN Outcompete the Wild-Type Form in Free-Living and Static Conditions but Not In Planta [Environmental Microbiology]]]> Members of the genus Burkholderia colonize diverse ecological niches. Among the plant-associated strains, Paraburkholderia phytofirmans PsJN is an endophyte with a broad host range. In a spatially structured environment (unshaken broth cultures), biofilm-constructing specialists of P. phytofirmans PsJN colonizing the air-liquid interface arose at high frequency. In addition to forming a robust biofilm in vitro and in planta on Arabidopsis roots, those mucoid phenotypic variants display a reduced swimming ability and modulate the expression of several microbe-associated molecular patterns (MAMPs), including exopolysaccharides (EPS), flagellin, and GroEL. Interestingly, the variants induce low PR1 and PDF1.2 expression compared to that of the parental strain, suggesting a possible evasion of plant host immunity. We further demonstrated that switching from the planktonic to the sessile form did not involve quorum-sensing genes but arose from spontaneous mutations in two genes belonging to an iron-sulfur cluster: hscA (encoding a cochaperone protein) and iscS (encoding a cysteine desulfurase). A mutational approach validated the implication of these two genes in the appearance of variants. We showed for the first time that in a heterogeneous environment, P. phytofirmans strain PsJN is able to rapidly diversify and coexpress a variant that outcompete the wild-type form in free-living and static conditions but not in planta.

IMPORTANCE Paraburkholderia phytofirmans strain PsJN is a well-studied plant-associated bacterium known to induce resistance against biotic and abiotic stresses. In this work, we described the spontaneous appearance of mucoid variants in PsJN from static cultures. We showed that the conversion from the wild-type (WT) form to variants (V) correlates with an overproduction of EPS, an enhanced ability to form biofilm in vitro and in planta, and a reduced swimming motility. Our results revealed also that these phenotypes are in part associated with spontaneous mutations in an iron-sulfur cluster. Overall, the data provided here allow a better understanding of the adaptive mechanisms likely developed by P. phytofirmans PsJN in a heterogeneous environment.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.02670-18 hwp:master-id:aem;AEM.02670-18 American Society for Microbiology 2019-06-01 Environmental Microbiology 85 11 e02670-18 e02670-18 hw_mjid:aem;85/11/e02670-18
<![CDATA[Comparative Metagenomics Reveals Enhanced Nutrient Cycling Potential after 2 Years of Biochar Amendment in a Tropical Oxisol [Environmental Microbiology]]]> The complex structural and functional responses of agricultural soil microbial communities to the addition of carbonaceous compounds such as biochar remain poorly understood. This severely limits the predictive ability for both the potential enhancement of soil fertility and greenhouse gas mitigation. In this study, we utilized shotgun metagenomics in order to decipher changes in the microbial community in soil microcosms after 14 days of incubation at 23°C, which contained soils from biochar-amended and control plots cultivated with Napier grass. Our analyses revealed that biochar-amended soil microbiomes exhibited significant shifts in both community composition and predicted metabolism. Key metabolic pathways related to carbon turnover, such as the utilization of plant-derived carbohydrates as well as denitrification, were enriched under biochar amendment. These community shifts were in part associated with increased soil carbon, such as labile and aromatic carbon compounds, which was likely stimulated by the increased available nutrients associated with biochar amendment. These findings indicate that the soil microbiome response to the combination of biochar addition and to incubation conditions confers enhanced nutrient cycling and a small decrease in CO2 emissions and potentially mitigates nitrous oxide emissions.

IMPORTANCE The incorporation of biochar into soil is a promising management strategy for sustainable agriculture owing to its potential to sequester carbon and improve soil fertility. Expanding the addition of biochar to large-scale agriculture hinges on its lasting beneficial effects on the microbial community. However, there exists a significant knowledge gap regarding the specific role that biochar plays in altering the key biological soil processes that influence plant growth and carbon storage in soil. Previous studies that examined the soil microbiome under biochar amendment principally characterized only how the composition alters in response to biochar amendment. In the present study, we shed light on the functional alterations of the microbial community response 2 years after biochar amendment. Our results show that biochar increased the abundance of genes involved in denitrification and carbon turnover and that biochar-amended soil microcosms had a reduction in cumulative CO2 production.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.02957-18 hwp:master-id:aem;AEM.02957-18 American Society for Microbiology 2019-06-01 Environmental Microbiology 85 11 e02957-18 e02957-18 hw_mjid:aem;85/11/e02957-18
<![CDATA[Multilocus Sequence Analysis, a Rapid and Accurate Tool for Taxonomic Classification, Evolutionary Relationship Determination, and Population Biology Studies of the Genus Shewanella [Methods]]]> The genus Shewanella comprises a group of marine-dwelling species with worldwide distribution. Several species are regarded as causative agents of food spoilage and opportunistic pathogens of human diseases. In this study, a standard multilocus sequence analysis (MLSA) based on six protein-coding genes (gyrA, gyrB, infB, recN, rpoA, and topA) was established as a rapid and accurate identification tool in 59 Shewanella type strains. This method yielded sufficient resolving power in regard to enough informative sites, adequate sequence divergences, and distinct interspecies branches. The stability of phylogenetic topology was supported by high bootstrap values and concordance with different methods. The reliability of the MLSA scheme was further validated by identical phylogenies and high correlations of genomes. The MLSA approach provided a robust system to exhibit evolutionary relationships in the Shewanella genus. The split network tree proposed twelve distinct monophyletic clades with identical G+C contents and high genetic similarities. A total of 86 tested strains were investigated to explore the population biology of the Shewanella genus in China. The most prevalent Shewanella species was Shewanella algae, followed by Shewanella xiamenensis, Shewanella chilikensis, Shewanella indica, Shewanella seohaensis, and Shewanella carassii. The strains frequently isolated from clinical and food samples highlighted the importance of increasing the surveillance of Shewanella species. Based on the combined genetic, genomic, and phenotypic analyses, Shewanella upenei should be considered a synonym of S. algae, and Shewanella pacifica should be reclassified as a synonym of Shewanella japonica.

IMPORTANCE The MLSA scheme based on six housekeeping genes (HKGs) (gyrA, gyrB, infB, recN, rpoA, and topA) is well established as a reliable tool for taxonomic, evolutionary, and population diversity analyses of the genus Shewanella in this study. The standard MLSA method allows researchers to make rapid, economical, and precise identification of Shewanella strains. The robust phylogenetic network of MLSA provides profound insight into the evolutionary structure of the genus Shewanella. The population genetics of Shewanella species determined by the MLSA approach plays a pivotal role in clinical diagnosis and routine monitoring. Further studies on remaining species and genomic analysis will enhance a more comprehensive understanding of the microbial systematics, phylogenetic relationships, and ecological status of the genus Shewanella.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.03126-18 hwp:master-id:aem;AEM.03126-18 American Society for Microbiology 2019-06-01 Methods 85 11 e03126-18 e03126-18 hw_mjid:aem;85/11/e03126-18
<![CDATA[Composition and Origin of the Fermentation Microbiota of Mahewu, a Zimbabwean Fermented Cereal Beverage [Food Microbiology]]]> Mahewu is a fermented cereal beverage produced in Zimbabwe. This study determined the composition and origin of mahewu microbiota. The microbiota of mahewu samples consisted of 3 to 7 dominant strains of lactobacilli and two strains of yeasts. Enterobacteriaceae were not detected. Candida glabrata was present in high cell counts from samples collected in summer but not from samples collected in winter. Millet malt is the only raw ingredient used in the production of mahewu and is a likely source of fermentation microbiota; therefore, malt microbiota was also analyzed by culture-dependent and high-throughput 16S rRNA gene sequencing methodologies. Millet malt contained 8 to 19 strains of Enterobacteriaceae, lactobacilli, bacilli, and very few yeasts. Strain-specific quantitative PCR assays were established on the basis of the genome sequences of Lactobacillus fermentum FUA3588 and FUA3589 and Lactobacillus plantarum FUA3590 to obtain a direct assessment of the identity of strains from malt and mahewu. L. fermentum FUA3588 and FUA3589 were detected in millet malt, demonstrating that millet malt is a main source of mahewu microbiota. Strains which were detected in summer were not detected in samples produced at the same site in winter. Model mahewu fermentations conducted with a 5-strain inoculum consisting of lactobacilli, Klebsiella pneumoniae, and Cronobacter sakazakii demonstrated that lactobacilli outcompete Enterobacteriaceae, which sharply decreased in the first 24 h. In conclusion, mahewu microbiota is mainly derived from millet malt microbiota, but minor components of malt microbiota rapidly outcompete Enterobacteriaceae and Bacillus species during fermentation.

IMPORTANCE This study provides insight into the composition and origin of the microbiota of mahewu and the composition of millet malt microbiota. Fermentation microbiota are often hypothesized to be derived from the environment, but the evidence remains inconclusive. Our findings confirm that millet malt is the major source of mahewu microbiota. By complementing culture methods with high-throughput sequencing of 16S rRNA amplicons and strain-specific quantitative PCR, this study provides evidence about the source of mahewu microbiota, which can inform the development of starter cultures for mahewu production. The study also documents the fate of Enterobacteriaceae during the fermentation of mahewu. There are concerns regarding the safety of traditionally prepared mahewu, and this requires in-depth knowledge of the fermentation process. Therefore, this study elucidated millet malt microbiota and identified cultures that are able to control the high numbers of Enterobacteriaceae that are initially present in mahewu fermentations.

2019-05-16T08:01:04-07:00 info:doi/10.1128/AEM.03130-18 hwp:master-id:aem;AEM.03130-18 American Society for Microbiology 2019-06-01 Food Microbiology 85 11 e03130-18 e03130-18 hw_mjid:aem;85/11/e03130-18


Average UCAS tariff point scores of first year, first degree students with highest qualification on entry of A-level or equivalent, excluding students on a foundation year. UCAS ENTRY POINTS 306

The ratio of female vs male students FEMALE TO MALE % 51:49

Average expenditure by department divided by number of students in that department, using a three year average expenditure (2007-2010) to allow for fluctuations. AVERAGE SPEND £4,734

Total number of first degree students. NUMBER OF STUDENTS 9,757


"Nous fouillons dans nos poches."

Translation:We are searching our pockets.

April 9, 2018

Quelle est la différence entre 'chercher' et 'fouiller'? Aussi, est-ce que c'est un mot a dire 'to find' autre que 'trouver' pour après on cherche ou fouille un chose?

April 9, 2018

chercher is a transitive verb whereas fouiller can be used transitively or intransitively

chercher means to look for (Je cherche mes clés. - I'm looking for my keys).

chercher can also mean to look up (such as to look up something in a dictionary)

fouiller when used transitively means to search (as in when a security guard searches you to determine if you have a concealed weapon).

fouiller can also mean to rummage when used intransitively

so consider these two sentences

Il a fouillé dans mon sac - He searched my bag (the security guard searched my bag to see if there was anything untoward hidden in it)

j'ai cherché mon sac - I searched for my bag (which I had mislaid)

April 9, 2018

Can someone explain how this relates to transportation, please?

June 20, 2018

When at an airport, you need to remove all items from your pockets before going through the security.

August 1, 2018

I thought you were looking for that special cigarette that sends you on a trip!

March 23, 2019
Learn French in just 5 minutes a day. For free.


IELTS: Describe a situation when you had to be nice (polite) to someone you dislike

IELTS results 2018

Here's an example of a question from Part 2 of the IELTS Speaking Test (with a sample answer).

This question asks about a situation in which you had to be nice (polite) to a person you didn't like.

The important words in this cue card are: situation, had to (obligation), polite/nice, and dislike.

In similar cue cards, the wording might be: a time when you had to be polite to someone, a person who was polite (nice) to you.

The sample answer gives an idea of how you could respond to this type of question about 'being polite'.

Obviously there are many different ways to answer it - depending on your own experience and knowledge.

IELTS Speaking Test Part 2 Cue Card Topic Questions

Describe a situation when you had to be nice (polite) to someone you dislike.

You should say:
  • who this person was
  • why you didn't like him/her
  • why you still had to be nice/polite to him/her?
and explain how you felt about the situation.

IELTS Speaking Test Answer | A time you had to be polite to someone you dislike

I can remember one occasion when I had to work as part of a team for a group project last semester at university.

In our group there was one person who I disliked (a little) because he was always trying to force his opinions on the other members of the group and wasn't very interested in hearing the ideas and views of the other people in the group.

The idea of group work is that everybody can make a contribution for the overall benefit of the group and not just follow the person who is most opinionated or bossy.

Anyway, we had to work together over a period of about four months and it was difficult at times because of his personality.

However, it was important for the group to develop the project as part our coursework and therefore it was necessary for us to all work together and try to maintain a good working relationship even though it was strained at times.

Although it was difficult at first because of his personality and the fact that he always thought that he was right and had the best ideas, eventually we all managed to work as a team and by the end of the project he wasn't really as bad as he had been at the beginning and was more willing to collaborate with the rest of us in the group.

And that was my experience of being nice or polite to someone I disliked.


EFCC begins manhunt for Diezani’s son

The Economic and Financial Crimes Commission has launched a manhunt for Ugonna, son to the former Minister of Petroleum Resources, Diezani Alison-Madueke. This is in connection with the alleged disbursement of money to INEC officials and election monitors in the build-up to the presidential election, Punchng report.

EFCC officials had stated that Diezani’s son served as a middleman between the ex-minister and the bank MD.

A senior official, who spoke to our correspondent on condition of anonymity, said, “We have launched a manhunt for Ugonna, who the suspects said was the one who drew up a list of individuals, who should be given the N23bn ($115m).

“We don’t know exactly where he is but we will get him. He needs to explain his role in the sharing of the money because the bank officials have been mentioning his name since they were brought in.”

Ugonna is the Head of Business Development and Commercial Marketing at Radam Maritime Services Limited, a firm, which is headed by his father, Alison Madueke, a former Chief of naval Staff, who is also under investigation by the EFCC for alleged money laundering.

He is said to have business interests in the United States and the United Kingdom.

Ugonna, who is an engineer, burst into the limelight in 2012 when photos showing his ostentatious lifestyle went viral on the Internet.

In one of such pictures, the graduate of University of Maryland College Park in the US, was seen flying in a private jet and later drinking champagne in a limousine. He has also been seen displaying wads of dollar notes on social media.


Altering the truth: The hidden world

Subject: Altering the truth: The hidden world
Date: 13 Mar 2019

Do you remember when you were a kid and you believed everything your parents told you? When Santa Clause, the Easter Bunny, and the Tooth Fairy were all real? Well, today there are still people who reside in this state, but not with their parents, but rather their government. In countries such as North Korea, Eritrea, and Saudi Arabia have censored media and little to no access to the truth ( It isn’t safe to be told lies and not the truth. Media should be free and not restricted, and everyone should have freedom of the press, as to not take away one’s sense of liberty.

When you take into account all of the reasons why withholding information and altering the truth from the people is wrong, it becomes a much bigger issue. First of all, the total population estimate in only the top 10 most censored countries is about 2,180,029,200 people which is about 28% of the entire world population ( In our lives, that’s like when your teacher tells each person their score on a test. If your class has 30 people in it, that means that 9 people would be told an incorrect score, either higher or lower than what they actually got. That wouldn’t be fair for those people because it wouldn’t be an accurate representation of what they know, and they won’t know what they need to review in the future or what they do well on. That inaccurate representation is what more than 28% of our world has to live with their entire lives. They don’t know what they need to look out for or what’s actually going on in the world, or even their own country. In most of the top 10 countries, the media is filled with propaganda and loving articles of their dearest leader ( When a country’s citizens are oblivious to the world around them, they can’t make good, educated decisions for themselves, taking away their personal liberties.

Of course, you might make the argument that it is beneficial to have a huge group of people that follow their leader blindly, and there are some good reasonings behind that such as unity or that people will be calm and not confused and they can just go about their lives ready to defend their country at any second. However, the fact that there isn’t resistance to the leader is a problem. When a leader doesn’t have anyone to keep them from making bad or foolish decisions. Without the people voting on an issue, or at least people in legislature/ Judicial systems keeping the balance in check, these bad decisions are going to go through, eventually ruining the country. Also, these tyrant rulers lie to the people to gain their trust and ensure their cooperation. According to, “dictators employ propaganda to ensure and sustain their public support”. The basic definition of propaganda is information of a misleading nature used to promote a particular political view. This shows that dictators lie to get support and cooperation from their people. I’m not sure about you, but I don’t want to blindly follow someone wo is going to lie to me. Therefore, it isn’t good to have people recklessly follow their leader.

When we are faced with problems, we shouldn’t avoid them, we have to find solutions. In many ways, these people are being lied to. This is an unjust tactic used by many leaders to avoid opposition. Would you want to be lied to? When other people face terrible issues such as these, we need to take action to fix this wrong and make it right again.
Works Cited
Britannica, The Editors of Encyclopaedia and John M. Cunningham. Dictatorship. 2019. March 2019. .
Current World Population. 5 March 2019. March 2019. .
ejc. kejncijned. kjencine, ejncejnt.
Guide, Ben's. Checks and Balances. n.d. March 2019. .
Staff, CPJ. 10 Most Censored Countries. 2015. March 2019. .


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I learned that I can contact Bloomberg Help Desk and got an answer from there: YAS_RISK is DV01/100, where DV01 is the dollar price change resulting from a one-basis-point change in yield. YAS_RISK is given in the currency of the bond, because DV01/100 is a percentage of the face value, and the face value currency is the currency of the bond. Please ...

I think it might be helpful to give an example here. So lets say you would want to estimate your PnL for a bond using the yield change and use YAS_RISK to retrieve your DV01 for a specific nominal. Assuming nominal of 1,000,000 Yield change: Lets use a realtime field for yield change RT_YLD_CHG_NET_1D This is in percentage To get the DV01 for your nominal ...

I've only got the same documentation as you but I'd say YAS_RISK is a rate of change per 100 bp interest rate change, i.e. the unit is percent which makes sense given that most bonds are quoted in percent on par. Change in interest rates refers to the consensus benchmark rates at comparable duration (by default). Details can be set up using YASD <GO>....

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Overlay personal big

Personal Server sign.

Personal Servers was a feature used on Roblox that allowed players to usually build whatever they want in-game and have it save on its own. Lots of players were able to join and help build with the owner of the game. Some restrictions though were applied such as who can visit, who can build, and who is banned. Oftentimes, personal servers were made mostly of terrain, with few non-terrain blocks rarely used for anything other than functionality.

The newest personal server icon.

Users must have been subscribed to a Builders Club membership tier before making a Personal Server. However, all players were able to join a personal server. When the place owner's Builders Club expires, the server is still kept, but another server cannot be made and if the place is changed into a regular game, it cannot be made a Personal Server until the owner got Builders Club again.

Personal servers were discontinued on June 8, 2016, according to a message from ROBLOX.

ROBLOX RALLY-Three ROBLOX administrators introduce personal servers at the 2011 ROBLOX Rally

ROBLOX RALLY-Three ROBLOX administrators introduce personal servers at the 2011 ROBLOX Rally.

Personal servers were first introduced by Njay, ostrichSized, and gen2integri at the 2011 ROBLOX Rally. The feature was fully released on November 2, 2011.[1] On May 26, 2016, it was discontinued to be replaced with Team Create.

ROBLOX describes a personal server as "a game that never closes."[2] Unlike a regular game place, when a server closes when all players have left, a personal server never closes. Therefore, the personal server never reverts to the way it was before users started playing it; this creates the opportunity for personal servers to be used as collaborative building projects. Personal servers can be changed back to a game place and vice versa.

The following is a chart explaining the differences between a personal server and a game place:

Personal ServerGame Place
Single Instance
Public by Default
Private by Default
Access Privileges
Ban List
Gear Allowed
Tickets Awarded for Visitors Before Removal
Builders Club to Create
Free to Play

Access Levels

How to promote/demote a user within a personal server

  1. The owner and administrators can assign access levels to specific users via clicking the player's name on the leaderboard. These access levels dictate what permissions that player has and what he can and cannot do within the personal server.

The following is a chart explaining the certain permissions various access levels have within a personal server:

Ability to play game
Ability to use build tools
Ability to promote/demote other users*
Ability to add and remove administrators

*Except promotion/demotion of administrators

It is advised to use caution when promoting players. Any changes they make to the server, whether constructive or destructive, will be automatically saved, unless the place's version has been changed to another non-destructed version.

Personal Servers within Groups

Personal Servers can be associated with particular groups. A feature of personal servers allows membership and admin can be awarded to members of a group with a certain rank or above. Like associating a place with a group, associating a personal server with a group makes that personal server appear on the group's page.

Personal Servers and the unique gameplay culture

In many Personal Servers, communities would form which would create a roblox group to further assign and divide the permissions of players.

External tools like F3X or a special custom build tool which were easily added by inserting them into game studio posed as alternatives to the traditional build tools.

However, there were stamper tool enthusiasts as well. This was a relatively more obscure community. They built mainly using the stamper tool feature enjoying its simple nature. They often employed the wiring present in the stamper tool to build devices that ran on inputs and outputs. As an example, float pads were often employed to power devices ranging from simple rockets to more complex mechanisms such as multi-floor elevators and claw machines.

These groups would make cities and active hubs to chat and play together with friends and create a social hierarchy, found nowhere else in all of Roblox. Since a large amount of people could get together in one server, it would often create an online city which was inhabited by active members which was able to continuously develop.

Criticisms and Issues

An issue with Personal Servers were griefers. This often occured in Personal Servers in which building rights were handed out too freely. Griefers would sabotage a place by flooding it with water or blowing up structures using C4 and explosive gear. This was easily remedied by removing the building rights of the person and reverting back to an earlier version before the griefing.

Personal Servers were often claimed to be a useless feature saying that many types of games on Personal Servers were also done in normal places. Many types of games such as, quiz games, RP games, and minigame places, were often run on Personal Servers due to the ease of setting such games up on the feature. These places often relied on hosts interacting with the other players, while many regular places automated the processes using scripts. This argument ignores the fact that the host interaction is often what set Personal Servers apart.

The stamper tools that came with Personal Servers were also prone to develop bugs overtime. There are examples such as, cloning a brick connected to a larger structure would cause the structure to erratically shift and deform, moving a brick connecting to a larger structure using the 4x4 movement option would do something similar to the clone tool problem, structures built using the basic building blocks would start to develop strange cracks and slowly shift as more was added to the structure, and the wiring tool and configure tool were prone to no longer work in servers with many bricks forcing a reset. These issues were completely ignored by Roblox who most likely wanted to deemphasize the Personal Server feature and emphasize normal places.

Team Create

Main Article: Roblox Studio

Team Create is a similar to Personal Servers, in that it allows collaborative building, except players are restricted to building in Roblox Studio instead of being able to do so in-game along with their avatars, and players need to invite others to build with them. Team Create was first announced in a blog post on April 13, 2016, while it was in Beta. It is released. Here are several images of Team Create:

Differences between Personal Servers and Team Create

  • On Personal Servers, players had access to the stamper tool and whichever other tool inside their inventory because their avatar was present, while on team create players do not have access to their avatar.
  • Personal Servers had access levels restricted to different players, but team create does not.
  • Personal Servers allowed the users to build in-game with their avatar instead of in studio like Team Create.
  • Personal Servers had an extra megaphone-like tool that would create thumbnails, like a camera, but this feature isn't available on Team Create.
  • While every player can join a Personal Server (except players who are banned from the server), players must be invited to a Team Create.

  1. ROBLOX Blog, Your Own Personal Server, Version 1.0,
  2. ROBLOX, What is a Personal Server?,


18:30 GMT +323 May 2019
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    Does this mean the resurgence of US shale oil production after two years of desolation will result in another crude price bust, similar to the one seen in late 2014? It does not: this time around, the market structure is different.

    The US remains one of the world’s major oil consumers, and the total volume of US oil consumption still exceeds any projected level of oil extraction in North America. In 2016, the US consumed an average of 19.63 mln bpd, and the demand has increased this year, as suggested by recent declines in US crude stockpiles. At this point, however, US oil production stands at 9.1 mln bpd.

    Moreover, with the construction of the Dakota Access Pipeline (DAPL) and Keystone XL, the lion’s share of US shale oil will flow to the refineries of the Gulf Coast, most notably, in Louisiana and Texas. US East Coast refiners and consumers, therefore, will be increasingly reliant on crude imports.

    "It should be somewhat supportive of (US oil prices) in the short run, particularly if the exports keep up. But it obviously is a challenge for the global market and a renewed threat to OPEC and their designs of keeping prices up," John Kilduff of Again Capital said.

    Atlantic Oil drilling
    The US and world crude prices are heading for parity, and in the medium-term, US oil prices are projected to outpace the price of the London-traded global Brent benchmark. This is partially because many US crude producers will find it more profitable to export their oil rather than selling it in the domestic market, especially after the Trump administration cuts US export customs tariffs.

    Even though at this point the US crude production might be holding back the gains in global oil prices resulting from the OPEC extraction cuts, in the medium-term, global oil prices are likely to settle at higher levels than they are now.

    Meanwhile, OPEC and other major oil producers are eyeing extending oil production caps till the first quarter of 2018. The decision is expected to be made during the OPEC meeting in Vienna on May 25.

    “We think we have everybody on board,” Al-Falih said. “Everybody I’ve talked to indicated that nine months was a wise decision.”

    Subsequently, oil prices advanced to a one-month high on the news, with Brent benchmark having gained 2.1 percent to $53.61/bbl, and US oil appreciating to $50.33/bbl. In the near-term, global oil prices are set to advance to $55-60/bbl, even though US shale production has increased by 10 percent since mid-2016, mainly because the global demand for fuel has also picked up in line with more robust economic growth in the advanced and developing economies alike.

    “OECD and non-OECD floating storage has been offloaded into tanks and consumed. We’re coming into high demand season, so I think everything is coming together with a consensus among producers, demand rising, and the global economy doing well; Japan, Europe and the US are all doing reasonably well economically. So we’re fairly optimistic,” Al-Falih said.

    Considering all the factors that drive the global oil market, US oil prices are likely to post more significant increases compared to gains in the Brent benchmark price. The extension and possible deepening of OPEC production cuts might not produce a massive oil rally, as the market will be levelled off by the US exports, but stronger global demand will contribute to easing the oil supply glut, with oil prices stabilizing at 5-10 percent higher than their present level.

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Mastery Mastership Masterpiece Mastermind Masterly Masterfully Masterful Master Sergeant Masthead Mastic Masticate Mastication Mastiff Mastitis Mastoiditis Masturbate Masturbation Masturbator Mat Matador

Masthead   Meaning in Urdu

1. Masthead : سر ورق - رسالے کا پہلا صفحہ : (noun) the title of a newspaper or magazine; usually printed on the front page and on the editorial page.

Title - the name of a work of art or literary composition etc..

Useful Words

Column - Editorial - Newspaper Column : مضمون : an article giving opinions or perspectives.

Front - Presence : سامنے : the immediate proximity of someone or something. "Get out of my front"

Entitle - Title : عنوان دینا : give a title to.

واش روم جانا ہے


Innovation and Divergence – A Division of Labor REMIX

The balance point between convergent/divergent processes marks the opportunity for innovative thinking.

Second Model of Agility and Divergence

Smith, however, highlights another important element within the division of labor: dexterity (aptitude, from within or without the process). In referring to dexterity, Smith attributes a creative aspect, which by nature is a divergent impulse. Although a company may be driven by a convergent and collective process (strategy, protocols, job functions, etc.), the individuals who make up a workforce are rather distributive in nature (distribution, in this case, defined as “branching” out; termination of a ramifying structure).

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Dexterity is both divergent and distributive, as it opens up the potential for invention/innovation within a given task, not only from the creative powers of a task-dedicated “specialist,” but also from the ingenuity of an industry outsider who can combine “the powers of the most distant or dissimilar objects.” (His example of the machine maker who develops a new technology for semi-related industry, or the boy who inadvertently develops a steam engine component differs from the example of the philosopher/speculator shedding light to an industry from an outside perspective). The act of re-interpreting and reinventing a process, making innovative leaps in technology creation, or combining “distant or dissimilar objects” from an outsider’s perspective—these actions exhibit a branching out (distribution) beyond organizational efficiency and convergence, and toward a more agile and experimental enterprise.

The first model (efficiency and convergence) presents an image wherein two people (1+1) operating the same task make up a quantitative set (one = one), whereas in the second model, we have a qualitative set where one may be greater than one, or two, or three or more at different points in time relative to the task at hand and the worker’s skill sets. The first model finds its gravitational pull toward the collective territories of “company,” whereas the second model finds its dispersive push toward the distributive pathways of human action.

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Sherif is the founder of KRAFT studio (for Kinematic, Robotic, Architectonic and Fabrication Technologies).

His responsibilities include the development of the computer aided architectural design (CAAD) curriculum within the department, besides managing the digital fabrication laboratory.


testsuite::test_iaf_dc - sli script for test of iaf_psc_alpha with external DC current
(test_iaf_dc) run -> compare cout with comments in file

test_iaf_dc.sli is a test of a specific feature of the iaf_psc_alpha
model. It is tested at which time and how an external DC current affects
the state of the neuron, the membrane potential in particular.

This is probably the simplest setup demonstrating how an external
continuous function (the DC) can be incorporated into a system with
point event interaction. A general introduction, including examples
not restricted to piecewise constant functions, can be found in [1].

The expected output is documented and briefly commented at the end of
the script.

Cleary, the result of this script is not resolution independent. The
choice of the computation step size determines at which point in time
the current affects the neuron.

There are 2 problems with this script:
1. A network element (here the DC generator) can only change its state
when it performs a computation (update) step t -> t + h.
Thus, the earliest possible time the DC generator can be switched on
is at t=h. This is the default. Therefore, the time of onset depends
on the computation step size.
2. The default delay between the DC generator and the neuron is the minimal
allowed value of 1 h. Therefore, the delay of the current depends on
the computation step size.

A sequence of further test scripts (SeeAlso key below) demonstrates and
verifies how the SLI script can be made independent of the computation
step size. This leads to properly aligned and identical voltage traces
for simulations carried out with different computation step sizes.

systems with applications to neuronal modeling. Biologial Cybernetics

Author: February 2004, Diesmann, Gewaltig, Plesser
SeeAlso: Source:


What is Conservation of Momentum – Definition

What is the Law of Conservation of Momentum

law of conservation of energy - pendulum

Let us assume the one dimensional elastic collision of two billiard balls,, the ball A and the ball B. We assume the net external force on this system of two balls is zero—that is, the only significant forces during the collision are the forces that each ball exerts on the other. According to Newton’s third law, the two forces are always equal in magnitude and opposite in direction. Hence, the impulses that act on the two balls are equal and opposite, and the changes in momentum of the two balls are equal and opposite.

The law of conservation of linear momentum in words:

Nuclear and Reactor Physics:

Advanced Reactor Physics:

See above:

Laws of Conservation


Analyze the different approaches to innovation discussed in this chapter to determine which approach you think would be the greatest value to the greatest number of organizations. Explain your rationale.

Create one innovative approach that is not discussed in the textbook for increasing the amount of innovation within a large company. Describe this approach in detail and explain how companies could benefit from it.


How to use property as part of retirement savings

Multi generational family enjoying a barbecue outside while keeping wealth in the family

Many older people rely on rental property to supplement their income in retirement, and often want to pass this on to the next generation without them being hit by hefty tax charges.

Landlords have had to contend with several changes in recent years which have bumped up their tax costs before they even consider the potential Inheritance Tax (IHT) bills they might leave behind.

Try our free inheritance tax calculator now

For example, the amount of tax relief landlords can claim on mortgage interest payments is gradually being reduced. It used to be possible for landlords to claim tax relief on mortgage interest payments at their marginal rate. Since April 2017, however, this has begun to be cut back and by 2020 will be just 20% for all tax-paying landlords, regardless of whether they are basic, higher or additional rate taxpayers.

There’s also an additional 3% stamp duty surcharge, introduced in April 2016, which is payable each time a landlord buys a property costing more than £125,000 to add to their portfolios.

Tax bills don’t end when you die

Tax bills don’t stop there. Landlords who leave rental properties to loved ones in their wills risk leaving their beneficiaries with potentially steep Inheritance Tax (IHT) bills unless they plan carefully.

Under current rules, IHT is payable at a rate of 40% on the value of your estate above £325,000 subject to certain exemptions. That means if, for example, you have rental properties worth £600,000 which you want to leave to loved ones when you die, after taking account of the £325,000 allowance, your beneficiaries would have to hand £110,000 over to the taxman in IHT (40% of £275,000).

This may mean that beneficiaries have to sell the property or properties to pay for IHT bills, even if it was your intention that it these rental homes would hopefully provide them with both an income and capital growth in future.

Options for landlords wanting to pass on rental property when they die

One way for landlords to avoid leaving beneficiaries with hefty IHT bills is to consider gifting one or more rental properties whilst they are still alive. Provided you live for seven years after making the gift, there won’t be IHT for beneficiaries to pay when you die.

Bear in mind, however, that you’ll still have to pay Capital Gains Tax (CGT) on the difference between the cost of the property when you bought it and its value when you pass it to your beneficiaries.

There is an annual tax-free allowance of £11,700 this tax year which can be used before incurring a CGT charge. Landlords paying tax at the basic rate have to pay 18% CGT on anything in excess of this allowance, less expenses such as legal fees or stamp duty, rising to 28% for higher rate taxpayers.

The rules if properties are held in a limited company

Some landlords choose to hold their rental properties within a limited company. Limited companies aren’t affected by mortgage interest relief restrictions because they pay corporation tax on rental income rather than income tax.

However, there are often significant costs involved setting up a limited company to hold existing rental properties, as the company has to buy the properties, meaning the landlord must pay stamp duty and legal costs, as well as CGT depending on how much the properties are worth at the time.

It’s also important to remember that passing on shares in a limited company that holds buy-to-let properties isn’t IHT-free as business property relief, which exempts some businesses from IHT, doesn’t apply to companies which deals “wholly or mainly” with property, land, or stocks and shares.

Tax rules can be very complex and often change over time, so always seek expert professional advice on ways you might be able to minimise any potential IHT liability.

For expert planning advice in this area, you can request a free guide to inheritance tax from our long term partner St. James’s Place Wealth Management or take advantage of a no obligation financial review.

The value of an investment with St. James’s Place will be directly linked to the performance of selected funds and may fall as well as rise. You may get back less than the amount you invested.

The levels and bases of taxation, and reliefs from taxation, can change at any time and depend on individual circumstances.

Trusts are not regulated by the Financial Conduct Authority.


3.6 Plant Communication: Example 2

This week we continue our systematic review of a plant's sensory systems by exploring responses to volatile chemicals (in other words, what a plant smells). We start with an overview of the plant cell, briefly review human olfaction (smell), and then explore how fruits know when to ripen. From there we go over three different experiments that explore plant responses to volatile chemicals and start exploring the controversial question, "Do plants communicate with each other?".

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Abstract #2058

Axon Diameter Distribution (ADD) MRI

Raisa Z. Freidlin1, Evren Ozarslan2, Sinisa Pajevic1, Michal E. Komlosh3, Peter J. Basser3

1NIH/CIT/DCB, Bethesda, MD, United States; 2Radiology, BWH, Harvard Medical School, Boston, MA, United States; 3NIH/NICHD/PPITS, Bethesda, MD, United States

Obtained the axon diameter distribution (ADD) for fascicles in any orientation, anywhere within a brain volume, is based on estimate of an average propagator in each voxel using a 3-D generalization that determines the direction of maximum diffusivity and then estimates the DW signal attenuation profile.