Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.     

Diet and Geophagy Across a Western Amazonian Parrot Assemblage

We identified species‐ and community‐level dietary characteristics for a species‐rich Amazonian parrot assemblage to determine relationships among dietary metrics and use of geophagy sites. Previous studies suggest that soil is consumed at geophagy sites in this region mainly to supplement dietary sodium. We accumulated 1400 feeding records for 16 parrot species over 2 yr and found that seeds, flowers, and fruit pulp featured prominently in diets, while bark, insects, and lichen were consumed in small quantities. Food availability across 1819 trees was measured, and we found that flower availability was highest in the dry season and fruit production peaked in the wet season, but that phenology patterns of the 20 most commonly foraged plant species suggest no serious food bottlenecks. Partitioning of available food resources among the 13 most commonly encountered parrots is suggested by an ordination analysis (DCA), which placed the large macaws (Ara) with the Amazona parrots at the ‘primary forest’ end of a dietary resource axis and four smaller species at the ‘successional forest’ end of the axis. Parrot species associated with successional forest also consumed less plant species overall. Furthermore, these parrot species consuming successional forest resources had higher claylick visitation rates than those consuming primary forest resources suggesting they derive the greatest benefits from soil consumption.     

Towards an Informative Mutant Phenotype for Every Bacterial Gene

Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.     

Nitrogen or phosphorus limitation in rich fens? - Edaphic differences explain contrasting results in vegetation development after fertilization

Background and aims

Many rich fens are threatened by high nutrient inputs, but the literature is inconsistent with respect to the type of nutrient limitation and the influence of edaphic characteristics.

Methods

We performed experiments with N- and P-fertilization in three endangered rich fen types: floating fen with Scorpidium scorpioides, non-floating fen with Scorpidium cossonii, floodplain fen with Hamatocaulis vernicosus. In addition, K-fertilization was carried out in the floodplain fen.

Results

The floodplain fen showed no response to P-addition, but N- and K-addition led to grass encroachment and decline of moss cover and species richness. In contrast, in the P-limited floating fen with S. scorpioides, P-addition led to increased vascular plant production at the expense of moss cover. Scorpidium scorpioides, however, also declined after N-addition, presumably due to ammonium toxicity. The fen with S. cossonii took an intermediate position, with NP co-limitation. These striking contrasts corresponded with edaphic differences. The N-limited fen showed low Ca:Fe ratios and labile N-concentrations, and high concentrations of plant-available P and Fe-bound P. The P-limited fen showed an opposite pattern with high Ca:Fe ratios and labile N-concentrations, and low P-concentrations.

Conclusions

This implies that edaphic characteristics dictate the nature of nutrient limitation, and explain contrasting effects of N- and P-eutrophication in different fens.     

Structure of Clostridium difficile PilJ Exhibits Unprecedented Divergence from Known Type IV Pilins

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface.     

Four Children and Yale: The Making of a Human Geneticist: The Grover Powers Lecture 2014

Dr. Leon E. Rosenberg delivered the following presentation as the Grover Powers Lecturer on May 14, 2014, which served as the focal point of his return to his “adult home” as a Visiting Professor in the Department of Pediatrics. Grover F. Powers, MD, was one of the most influential figures in American Pediatrics and certainly the leader who created the modern Department of Pediatrics at Yale when he was recruited in 1921 from Johns Hopkins and then served as its second chairman from 1927 to 1951. Dr. Powers was an astute clinician and compassionate physician and fostered and shaped the careers of countless professors, chairs, and outstanding pediatricians throughout the country. This lectureship has continued yearly since it first honored Dr. Powers in 1956. The selection of Dr. Rosenberg for this honor recognizes his seminal role at Yale and throughout the world in the fostering and cultivating of the field of human genetics. Dr. Rosenberg served as the inaugural Chief of a joint Division of Medical Genetics in the Departments of Pediatrics and Internal Medicine; he became Chair when this attained Departmental status. Then he served as Dean of the Medical School from 1984 to 1991, before he became President of the Pharmaceutical Research Institute at Bristol-Myers Squibb and later Senior Molecular Biologist and Professor at Princeton University, until his recent retirement. Dr. Rosenberg has received numerous honors that include the Borden Award from the American Academy of Pediatrics, the McKusick Leadership Award from the American Society for Human Genetics, and election to the Institute of Medicine and the National Academy of Sciences.     

Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

In recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based on Bacteroidales 16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universal Bacteroidales marker (AllBac), including 66% of samples from cantaloupe farms (3.6 log₁₀ genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log₁₀ GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log₁₀ GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation between Bacteroidales and generic Escherichia coli across all samples. This study provides evidence that Bacteroidales markers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.     

OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis

Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.