Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts

Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces.     

Persisting Hyper-abundance of Leaf-cutting Ants (Atta spp.) at the Edge of an Old Atlantic Forest Fragment

Leaf-cutting ants (LCAs) profoundly benefit from edge creation in Neotropical forests, where they act as a keystone species and disturbance agent. In view of their poorly explored population dynamics, the question arises whether high densities of LCAs are a transitional or a persisting phenomenon. We studied the temporal variation of LCA colony densities at the edge of the Brazilian Atlantic forest. At physically stable edges of an old forest fragment, densities of Atta cephalotes and Atta sexdens (11 and five times higher in a 50 m edge zone in comparison with the forest interior) persisted over a 4-yr interval (2001–2005) with no significant difference in densities between years. Species-specific per colony growth rates ranged from 12 to −5 percent/yr, suggesting that populations were approximately at equilibrium. High rates of colony turnover (little less than 50% in 4 yr) indicated an average colony life span of about 7 yr—a life expectancy considerably lower than previous estimates for Atta colonies. Stable, hyper-abundant populations of LCAs accord with the constantly high availability of palatable pioneer vegetation (the preferred food source of LCAs) at forest edges and are expected to persist in time as long as forests are characterized by high edge to interior ratios, with potentially long-lasting consequences for the ecosystem.     

NanC Crystal Structure, a Model for Outer-Membrane Channels of the Acidic Sugar-Specific KdgM Porin Family

Sialic acids are acidic sugars present mostly on vertebrate cell surfaces, which can be metabolized by bacteria and act as an inflammation signal. N-Acetylneuraminic acid, the most abundant sialic acid, can enter into Escherichia coli K12 through NanC, an N-acetylneuraminic acid-inducible outer-membrane channel. With its 215 residues, NanC belongs to the family of small monomeric KdgM-related porins. KdgM homologues are found in gammaproteobacteria, including major plant and human pathogens, and together they define a large family of putative acidic sugar/oligosaccharide transporters, which are as yet poorly characterized. Here, we present the first high-resolution structure of a KdgM family member. NanC folds into a 28-Å-high, 12-stranded β-barrel, resembling the β-domain of autotransporter NalP and defining an open pore with an average radius of 3.3 Å. The channel is lined by two strings of basic residues facing each other across the pore, a feature that appears largely conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.     

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.     

Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin

Nearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold. Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasma-mass spectrometry showed that the iron in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation. Agronomic evaluation of the high-iron rice lines did not reveal a yield penalty or significant changes in trait characters, except for a tendency to earlier flowering. Overall, we have demonstrated that rice can be engineered with a small number of genes to achieve iron biofortification at a dietary significant level.     

Validation of a Nylon-Flocked-Swab Protocol for Efficient Recovery of Bacterial Spores from Smooth and Rough Surfaces

In order to meet planetary-protection requirements, culturable bacterial spore loads are measured representatively for the total microbial contamination of spacecraft. However, the National Aeronautics and Space Administration''s (NASA''s) cotton swab protocols for spore load determination have not changed for decades. To determine whether a more efficient alternative was available, a novel swab was evaluated for recovery of different Bacillus atrophaeus spore concentrations on stainless steel and other surfaces. Two protocols for the nylon-flocked swab (NFS) were validated and compared to the present NASA standard protocol. The results indicate that the novel swab protocols recover 3- to 4-fold more (45.4% and 49.0% recovery efficiency) B. atrophaeus spores than the NASA standard method (13.2%). Moreover, the nylon-flocked-swab protocols were superior in recovery efficiency for spores of seven different Bacillus species, including Bacillus anthracis Sterne (recovery efficiency, 20%). The recovery efficiencies for B. atrophaeus spores from different surfaces showed a variation from 5.9 to 62.0%, depending on the roughness of the surface analyzed. Direct inoculation of the swab resulted in a recovery rate of about 80%, consistent with the results of scanning electron micrographs that allowed detailed comparisons of the two swab types. The results of this investigation will significantly contribute to the cleanliness control of future life detection missions and will provide significant improvement in detection of B. anthracis contamination for law enforcement and security efforts.The recent discovery of liquid water on Mars has sparked debate about the possibility of extraterrestrial life (37). Consequently, highly sensitive biosensors will be deployed onboard spacecraft like the Mars Science Laboratory (MSL), using technologies such as gas chromatographical analysis to search for the smallest traces of life (http://mars.jpl.nasa.gov/msl/mission/). Contamination of equipment by terrestrial microorganisms resulting from a lack of spacecraft cleanliness could significantly compromise the integrity of life detection missions and result in falsely positive extraterrestrial life signals. The prevention of this so-called “forward contamination” is one major goal of American and European space agencies'' planetary-protection efforts. Regular determination of a spacecraft''s bioload and the mission components throughout assembly are mandatory for detecting unacceptably high contamination that exceeds levels set by the United Nations treaty (Outer Space Treaty [11]).Modern spacecraft hardware is very susceptible to standard heat sterilization protocols, so baking the entire spacecraft, such as the Viking Lander Capsule at 111.7°C ± 1.7°C for 23 to 30 h is no longer feasible (30). Alternative cleaning and sterilization methodologies for spacecraft components prior to assembly (i.e., nonthermal plasma technologies) have been discussed (36). However, after integration, sterile hardware is exposed to a significant risk of contamination during assembly, testing, and launching operations. Because of limited access to integrated spacecraft components, the microbial cleanliness of a spacecraft and its surroundings is meticulously maintained through frequent cleaning and sterilization routines. Therefore, the regular and frequent detection of possible contaminants in the assembly environment is more important than ever.To estimate the severity of microbial contamination, the National Aeronautics and Space Administration''s (NASA''s) standard procedure focuses on aerobic, mesophilic spores (26). Briefly, surface samples are taken from spacecraft using moist cotton swabs or wipes. After an extraction procedure, the samples are subjected to a short heat shock (15 min; 80°C) to kill vegetative cells and then pour plated in Trypticase soy agar (TSA) for the enumeration of CFU. This protocol was originally developed for the Viking mission more than 3 decades ago (30) and has remained, for the most part, unchanged.Recent studies have shown that cotton swabs have acceptable recovery efficiencies for Bacillus spores (41.7%) (32) but, due to their organic nature, may raise residue problems on surfaces. Furthermore, their comparatively high DNA content could lead to false positives or inhibition should NASA one day incorporate molecular technologies into their microbial-detection protocols (7).Based on these observations, researchers are beginning to move away from cotton in favor of alternative swabs made from rayon or macrofoam (6, 18). A recent study reported high recovery efficiencies for various vegetative cells from stainless steel surfaces by applying a novel swab with a bulb-shaped head flocked with nylon fibers (12). Patented in 2004, this design facilitates the release of particulates and microbes, resulting in a significantly higher detection rate. The broad applicability of these nylon-flocked swabs (NFS) has been demonstrated by their use in various clinical studies isolating pathogens from medical environments (1, 10, 20).General studies on surface-sampling tools have clearly shown that the swab material and the extraction method are the dominant factors in spore recovery efficiencies (32). Additionally, the properties of the surface to be sampled affect sample recovery (8). For planetary-protection applications, the broad variety of novel materials used in spacecraft construction must be considered. The Mars Exploration Rover mission craft, for example, was composed of at least five kinds of surface materials (http://marsrovers.jpl.nasa.gov/overview). While the cruise stage was constructed primarily of aluminum and the aeroshell consisted of aluminum honeycomb structures, the lander itself was made of titanium and graphite composite (carbon fiber-reinforced plastic [CFRP]). The airbag and the parachutes were made of Vectran and polyester/nylon fabrics. These different materials are quite challenging for sampling tools. Accurate sampling of materials with various surface textures will require planetary-protection programs to introduce novel swab materials.To our knowledge, no investigations have been performed to compare the recovery of spores from different spacecraft surfaces. Previous studies have compared cotton and synthetic sampling materials, but only on stainless steel surfaces (19), and no studies have compared sampling methods on actual spacecraft materials (7).Recently published protocols for spore detection have been based on one specific Bacillus species and/or on one type of surface. Unfortunately, these protocols provide no insight into the effects of varying these factors (4-6, 8, 9, 14, 18), as requested by USP (United States Pharmacopeia) 1223 for validation of alternative microbial methods (3). Some of the aforementioned studies were conducted in response to B. anthracis terrorism incidents in 2001 and used B. atrophaeus as a surrogate. Consequently, information about the actual sampling efficiency of B. anthracis spores is quite limited and may vary significantly from the B. atrophaeus data.In this comprehensive study, we evaluated the novel nylon-flocked swab and a corresponding protocol to recover Bacillus spores from five different spacecraft-related surfaces. It should be noted that although stainless steel served as the standard test surface, it is not a predominant material in spacecraft; however, since the majority of previous (sampling) studies were performed on stainless steel, it represents a universally recognized carrier and also serves as a conservative proxy for the average roughness of the materials used in space science.Our nylon-flocked-swab protocol was validated with respect to accuracy, precision, limit of detection, linearity, and robustness (3). Moreover, its specificity was determined by applying spores of seven different Bacillus species, including the avirulent, attenuated strain Bacillus anthracis Sterne, and by comparing the resulting recovery efficiencies. The results in this communication will significantly contribute to planetary-protection protocols and could also be of high interest for public health issues.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Between-year variation in seed weights across altitudes in the high-alpine plant <Emphasis Type="Italic">Eritrichium nanum</Emphasis>

Seed weight is a prominent life history trait of plants affecting dispersal, establishment, and survival. In alpine environments, the few studies investigating the effect of elevation on seed weight within species have mainly detected a decrease in seed weight with increasing elevation. This relationship is generally attributed to the adverse climate at high elevations. In order to test this hypothesis, we analyzed seed weight variation across altitudes (2,435–3,055 m a.s.l.) in two consecutive years that differed in weather conditions in the high-alpine cushion plant Eritrichium nanum. We found a significant reduction in seed weight with increasing elevation in both years, but in the growing season with more adverse weather conditions, the reduction was more substantial than in the more favorable year. We conclude that alpine plants may be able to produce well-developed seeds at low elevations in almost all years, independent of weather conditions, whereas reproduction through seeds is potentially limited to years of favorable weather at high elevation.