Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Isolation of tropomyosin particles from the cytosol of cultured cells and their protein composition analysis

The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.     

Counteractive biomass allocation responses to drought and damage in the perennial herb Convolvulus demissus

Herbivory and water shortage are key ecological factors affecting plant performance. While plant compensatory responses to herbivory include reallocation of biomass from below‐ground to above‐ground structures, plant responses to reduced soil moisture involve increased biomass allocation to roots and a reduction in the number and size of leaves. In a greenhouse study we evaluated the effects of experimental drought and leaf damage on biomass allocation in Convolvulus demissus (Convolvulaceae), a perennial herb distributed in central Chile, where it experiences summer drought typical of Mediterranean ecosystems and defoliation by leaf beetles and livestock. The number of leaves and internode length were unaffected by the experimental treatments. The rest of plant traits showed interaction of effects. We detected that drought counteracted some plant responses to damage. Thus, only in the control watering environment was it observed that damaged plants produced more stems, even after correcting for main stem length (index of architecture). In the cases of shoot : root ratio, relative shoot biomass and relative root biomass we found that the damage treatment counteracted plant responses to drought. Thus, while undamaged plants under water shortage showed a significant increase in root relative biomass and a significant reduction in both shoot : root ratio and relative shoot biomass, none of these responses to drought was observed in damaged plants. Total plant biomass increased in response to simulated herbivory, apparently due to greater shoot size, and in response to drought, presumably due to greater root size. However, damaged plants under experimental drought had the same total biomass as control plants. Overall, our results showed counteractive biomass allocation responses to drought and damage in C. demissus. Further research must address the fitness consequences under field conditions of the patterns found. This would be of particular importance because both current and expected climatic trends for central Chile indicate increased aridity.     

VPS13D bridges the ER to mitochondria and peroxisomes via Miro

Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER–mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson’s disease pathogenesis.     

Bristles before down: A new perspective on the functional origin of feathers

Over the course of the last two decades, the understanding of the early evolution of feathers in nonavian dinosaurs has been revolutionized. It is now recognized that early feathers had a simple form comparable in general structure to the hairs of mammals. Insight into the prevalence of simple feathers throughout the dinosaur family tree has gradually arisen in tandem with the growing evidence for endothermic dinosaur metabolisms. This has led to the generally accepted opinion that the early feather coats of dinosaurs functioned as thermo insulation. However, thermo insulation is often erroneously stated to be a likely functional explanation for the origin of feathers. The problem with this explanation is that, like mammalian hair, simple feathers could serve as insulation only when present in sufficiently high concentrations. The theory therefore necessitates the origination of feathers en masse. We advocate for a novel origin theory of feathers as bristles. Bristles are facial feathers common among modern birds that function like mammalian tactile whiskers, and are frequently simple and hair‐like in form. Bristles serve their role in low concentrations, and therefore offer a feasible first stage in feather evolution.     

Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in México

Background

Aedes aegypti, the ‘yellow fever mosquito’, is the primary vector to humans of dengue and yellow fever flaviviruses (DENV, YFV), and is a known vector of the chikungunya alphavirus (CV). Because vaccines are not yet available for DENV or CV or are inadequately distributed in developing countries (YFV), management of Ae. aegypti remains the primary option to prevent and control outbreaks of the diseases caused by these arboviruses. Permethrin is one of the most widely used active ingredients in insecticides for suppression of adult Ae. aegypti. In 2007, we documented a replacement mutation in codon 1,016 of the voltage-gated sodium channel gene (para) of Ae. aegypti that encodes an isoleucine rather than a valine and confers resistance to permethrin. Ile1,016 segregates as a recessive allele conferring knockdown resistance to homozygous mosquitoes at 5–10 µg of permethrin in bottle bioassays.

Methods and Findings

A total of 81 field collections containing 3,951 Ae. aegypti were made throughout México from 1996 to 2009. These mosquitoes were analyzed for the frequency of the Ile1,016 mutation using a melting-curve PCR assay. Dramatic increases in frequencies of Ile1,016 were recorded from the late 1990''s to 2006–2009 in several states including Nuevo León in the north, Veracruz on the central Atlantic coast, and Yucatán, Quintana Roo and Chiapas in the south. From 1996 to 2000, the overall frequency of Ile1,016 was 0.04% (95% confidence interval (CI95) = 0.12%; n = 1,359 mosquitoes examined). The earliest detection of Ile1,016 was in Nuevo Laredo on the U.S. border in 1997. By 2003–2004 the overall frequency of Ile1,016 had increased ∼100-fold to 2.7% (±0.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (±1.15% CI95; n = 473). This was followed in 2007–2009 by a sudden jump in Ile1,016 frequency to 33.2% (±1.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity in Ile1,016 frequencies among 2007–2008 collections, which ranged from 45.7% (±2.00% CI95) in the state of Veracruz to 51.2% (±4.36% CI95) in the Yucatán peninsula and 14.5% (±2.23% CI95) in and around Tapachula in the state of Chiapas. Spatial heterogeneity was also evident at smaller geographic scales. For example within the city of Chetumal, Quintana Roo, Ile1,016 frequencies varied from 38.3%–88.3%. A linear regression analysis based on seven collections from 2007 revealed that the frequency of Ile1,016 homozygotes accurately predicted knockdown rate for mosquitoes exposed to permethrin in a bioassay (R² = 0.98).

Conclusions

We have recorded a dramatic increase in the frequency of the Ile1,016 mutation in the voltage-gated sodium channel gene of Ae. aegypti in México from 1996 to 2009. This may be related to heavy use of permethrin-based insecticides in mosquito control programs. Spatial heterogeneity in Ile1,016 frequencies in 2007 and 2008 collections may reflect differences in selection pressure or in the initial frequency of Ile1,016. The rapid recent increase in Ile1,016 is predicted by a simple model of positive directional selection on a recessive allele. Unfortunately this model also predicts rapid fixation of Ile1,016 unless there is negative fitness associated with Ile1,016 in the absence of permethrin. If so, then spatial refugia of susceptible Ae. aegypti or rotational schedules of different classes of adulticides could be established to slow or prevent fixation of Ile1,016.