Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

Conserved axoneme symmetry altered by a component beta-tubulin

Ninefold microtubule symmetry of the eukaryotic basal body and motile axoneme has been long established [1-3]. In Drosophila, these organelles contain distinct but similar beta-tubulin isoforms [4-10]: basal bodies contain only beta1-tubulin, and only beta2-tubulin is used for assembly of sperm axonemes. A single alpha-tubulin functions throughout spermatogenesis [11,12]. Thus, differences in organelle assembly reside in beta-tubulin. We tested the ability of beta1 to function in axonemes and found that beta1 alone could not generate axonemes. Small sequence differences between the two isoforms therefore mediate large differences in assembly capacity, even though these two related organelles have a common evolutionarily ancient architecture. In males with equal beta1 and beta2, beta1 was co-incorporated at equimolar ratio into functional sperm axonemes. When beta1 exceeded beta2, however, axonemes with 10 doublets were produced, an alteration unprecedented in natural phylogeny. Addition of the tenth doublet occurred by a novel mechanism, bypassing the basal body. It has been assumed that the instructions for axoneme morphogenesis reside primarily in the basal body, which normally serves as the axonemal template. Our data reveal that beta-tubulin requirements for basal bodies and axonemes are distinct, and that key information for axoneme architecture resides in the axonemal beta-tubulin.     

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.     

A new heterospecific foraging association between the puddingwife wrasse,Halichoeres radiatus,and the bar jack,Caranx Tuber: evaluation of the foraging consequences

Synopsis The bar jack,Caranx Tuber, was commonly observed to follow individual puddingwife wrasses,Halichoeres radiatus, that were foraging on the substrate. Individuals of both species actively pursued the other to maintain these heterospecific foraging teams, were sometimes attracted to feeding acts initiated by team partners, and the foraging rates of teamed jacks and wrasses were positively correlated. Pilfering of food items was rare, suggesting little, if any, competition cost of this foraging association. The ratio of bites to search in teamed jacks was over three times that when solitary, and jacks were sometimes aggressive to conspecifics attempting to join their team, suggesting that the association is beneficial to the jacks. Both bite and search rates were higher in puddingwifes when teamed with a jack, indicating that the association also benefits the wrasses. Benefits to puddingwifes may be derived directly from attendants because wrasses were sometimes attracted to jack foraging acts. However, increased foraging in wrasses may also be a consequence of heightened motivation to feed owing to heterospecific social facilitation.     

Environmental impacts of hybrid and electric vehicles—a review

Purpose

A literature review is undertaken to understand how well existing studies of the environmental impacts of hybrid and electric vehicles (EV) address the full life cycle of these technologies. Results of studies are synthesized to compare the global warming potential (GWP) of different EV and internal combustion engine vehicle (ICEV) options. Other impacts are compared; however, data availability limits the extent to which this could be accomplished.

Method

We define what should be included in a complete, state-of-the-art environmental assessment of hybrid and electric vehicles considering components and life cycle stages, emission categories, impact categories, and resource use and compare the content of 51 environmental assessments of hybrid and electric vehicles to our definition. Impact assessment results associated with full life cycle inventories (LCI) are compared for GWP as well as emissions of other pollutants. GWP results by life cycle stage and key parameters are extracted and used to perform a meta-analysis quantifying the impacts of vehicle options.

Results

Few studies provide a full LCI for EVs together with assessment of multiple impacts. Research has focused on well to wheel studies comparing fossil fuel and electricity use as the use phase has been seen to dominate the life cycle of vehicles. Only very recently have studies begun to better address production impacts. Apart from batteries, very few studies provide transparent LCIs of other key EV drivetrain components. Estimates of EV energy use in the literature span a wide range, 0.10?C0.24?kWh/km. Similarly, battery and vehicle lifetime plays an important role in results, yet lifetime assumptions range between 150,000?C300,000?km. CO₂ and GWP are the most frequently reported results. Compiled results suggest the GWP of EVs powered by coal electricity falls between small and large conventional vehicles while EVs powered by natural gas or low-carbon energy sources perform better than the most efficient ICEVs. EV results in regions dependant on coal electricity demonstrated a trend toward increased SO _x emissions compared to fuel use by ICEVs.

Conclusions

Moving forward research should focus on providing consensus around a transparent inventory for production of electric vehicles, appropriate electricity grid mix assumptions, the implications of EV adoption on the existing grid, and means of comparing vehicle on the basis of common driving and charging patterns. Although EVs appear to demonstrate decreases in GWP compared to conventional ICEVs, high efficiency ICEVs and grid-independent hybrid electric vehicles perform better than EVs using coal-fired electricity.     

Presynaptic proteins involved in exocytosis inDrosophila melanogaster: A genetic analysis

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely uponin vitro evidence. Recently, genetic approaches in mice, C.elegans and the fruitfly,Drosophila melanagaster, have been used to dissect thein vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release inDrosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, makingDrosophila an ideal model system to further unravel the intricacies of synaptic transmission.     

The evolution of coexistence: Reciprocal adaptation promotes the assembly of a simple community

Species coexistence may result by chance when co‐occurring species do not strongly interact or it may be an evolutionary outcome of strongly interacting species adapting to each other. Although patterns like character displacement indicate that coexistence has often been an evolutionary outcome, it is unclear how often the evolution of coexistence represents adaptation in only one species or reciprocal adaptation among all interacting species. Here, we demonstrate a strong role for evolution in the coexistence of guppies and killifish in Trinidadian streams. We experimentally recreated the temporal stages in the invasion and establishment of guppies into communities that previously contained only killifish. We combined demographic responses of guppies and killifish with a size‐based integral projection model to calculate the fitness of the phenotypes of each species in each of the stages of community assembly. We show that guppies from locally adapted populations that are sympatric with killifish have higher fitness when paired with killifish than guppies from allopatric populations. This elevated fitness involves effects traceable to both guppy and killifish evolution. We discuss the implications of our results to the study of species coexistence and how it may be mediated through eco‐evolutionary feedbacks.     

Declining foliar and litter δ¹⁵N diverge from soil, epiphyte and input δ¹⁵N along a 120,000 yr temperate rainforest chronosequence

Patterns in the natural abundance of nitrogen (N) isotopes (1?N and 1?N) can help in the understanding of ecosystem processes along environmental gradients, because some processes fractionate against the heavier isotope. We measured δ1?N in many components of the Franz Josef soil chronosequence in New Zealand to see how each component varied along the sequence and within sites, and to see what this variation can tell us about how ecosystem processes such as N losses change with soil age. We analyzed δ1?N in foliage from 18 woody species, abscised leaves from seven woody species, three soil horizons, bryophytes, lichens, bulk deposition, and nodules from the N-fixing tree Coriaria arborea (Coriariaceae). Foliar δ1?N varied significantly across plant species. Foliage and bulk litter became 1?N-depleted as soil age increased. Soil N from organic and mineral horizons was significantly more 1?N-enriched than bulk litter N at each site. Increasing precipitation also decreased foliar and soil δ1?N. Comparing input and whole ecosystem δ1?N revealed limited evidence for net fractionation during N losses. These trends are consistent with some combination of increasing fractionation during plant N uptake, mycorrhizal transfer, within-plant processing, and soil decomposition as soils age.     

Evolution of E. coli on [U-13C]Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

¹³C-Metabolic flux analysis (¹³C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled ¹³C-glucose, a commonly used isotope for ¹³C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (¹³C) or unlabeled (¹²C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between ¹²C and ¹³C-glucose as a carbon source for E. coli growth.